Circadian rhythms are the organism’s physiological and behavioral strategies for coping with daily oscillations in environment conditions. Inputs such as light and temperature feed into a molecular clock via anatomical and molecular input pathways and reset it every day. Light is the dominant cue for entraining the molecular clock, but temperature is also a pervasive resetting signal in natural environments. Paradoxically, clocks must be semi-resistant to temperature: they should not hasten in warm summer months or lag in the winter cold (this is called temperature compensation), but they can synchronize to the daily rise and fall of temperature (temperature entrainment) (Pittendrigh, 1960). Not only can temperature entrain the clock, it also has a role in seasonal adaptation by affecting the phase of behavior (see for example Majercak et al., 1999).

Molecular circadian clocks in eukaryotes are made up of negative transcriptional feedback loops (Dunlap, 1999). In Drosophila, the transcription factors CLOCK (CLK) and CYCLE (CYC) bind to E-boxes in the promoters of the clock genes period (per) and timeless (tim) and activate their transcription. PER and TIM proteins accumulate in the cytoplasm where they heterodimerize and enter the nucleus to feedback and repress the activity of CLK and CYC and thus downregulate their own transcription (Hardin, 2011). This main loop is strengthened by a scaffolding of interlocked feedback loops involving the transcription factors vrille (vri), PAR domain protein 1 (Pdp1) and clockwork orange (cwo). Post-translational modifications are well-established mechanisms for adjusting the speed and timing of the clock (Tataroglu and Emery, 2015).

Increasing evidence indicates that post-transcriptional mechanisms controlling gene expression are also critical for the proper function of circadian clocks in many organisms. In Drosophila, the post-transcriptional regulation of per mRNA has been best studied. per mRNA stability changes as a function of time (So and Rosbash, 1997). In addition, per contains an intron in its 3’UTR (dmpi8) that is alternatively spliced depending on temperature and lighting conditions (Majercak et al., 1999; Majercak et al., 2004). On cold days, the spliced variant is favored, causing an advance in the accumulation of per transcript levels as well as an advance of the evening activity peak. This behavioral shift means that the fly is more active during the day when the temperature would be most tolerable in their natural environment. The temperature sensitivity of dmpi8 is due to the presence of weak non-canonical splice sites. However, the efficiency of the underlying baseline splicing is affected by four single nucleotide polymorphisms (SNPs) in the per 3’UTR that vary in natural populations and form two distinct haplotypes (Low et al., 2012; Cao and Edery, 2017). Also, while this splicing is temperature-sensitive in two Drosophila species that followed human migration, two species that remained in Africa lack temperature sensitivity of dmpi8 splicing, (Low et al., 2008). Furthermore, Zhang et al. (2018) recently demonstrated that the the trans-acting splicing factor B52 enhances dmpi8 splicing efficiency, and this effect is stronger with one of the two haplotypes. per is also regulated post-transcriptionally by the TWENTYFOUR-ATAXIN2 translational activation complex (Zhang et al., 2013; Lim et al., 2011; Lim and Allada, 2013a; Lee et al., 2017). This complex works by binding to per mRNA as well as the cap-binding complex and poly-A binding protein. This may enable more efficient translation by promoting circularization of the transcript. Interestingly, this mechanism appears to be required only in the circadian pacemaker neurons. Non-canonical translation initiation has also been implicated in the control of PER translation (Bradley et al., 2012). Regulation of PER protein translation has also been studied in mammals, with RBM4 being a critical regulator of mPER1 expression (Kojima et al., 2007). In flies however, the homolog of RBM4, LARK, regulates the translation of DBT, a PER kinase (Huang et al., 2014). miRNAs have emerged as important critical regulators of circadian rhythms in Drosophila and mammals, affecting the circadian pacemaker itself, as well as input and output pathways controlling rhythmic behavioral and physiological processes (Tataroglu and Emery, 2015; Lim and Allada, 2013b).

RNA-associated proteins (RAPs) include proteins that either bind directly or indirectly to RNAs. They mediate post-transcriptional regulation at every level. Many of these regulated events – including alternative splicing, splicing efficiency, mRNA stability, and translation – have been shown to function in molecular clocks. Thus, to obtain a broad view of the Drosophila circadian RAP landscape and its mechanism of action, we performed an RNAi screen targeting 364 of these proteins. This led us to discover a role for the splicing factor P-element somatic inhibitor (PSI) in regulating the pace of the molecular clock through alternative splicing of tim.

Our results identify a novel post-transcriptional regulator of the circadian clock: PSI. PSI is required for the proper pace of both brain and body clock, and for proper phase-relationship with ambient temperature cycles. When Psi is downregulated, the circadian pacemaker speeds up and behavior phase under temperature cycles is advanced by 3 hr, and these phenotypes appear to be predominantly caused by an abnormal tim splicing pattern. Indeed, the circadian period and behavior phase of flies that can only produce functional TIM protein from a transgene missing most introns is insensitive to Psi downregulation. We note however that cwo’s splicing pattern is also affected by Psi downregulation, and we did not study sgg splicing pattern, although it might also be controlled by PSI (Wang et al., 2016). We therefore cannot exclude a small contribution of non-tim splicing events to PSI downregulation phenotypes, or that in specific tissues these other splicing events play a greater role than in the brain.

Interestingly, Psi downregulation results in an increase in intron inclusion events that are favored under cold conditions (tim-sc and tim-cold), while an intron inclusion event favored under warm conditions is decreased (tim-M). However, the ability of tim splicing to respond to temperature changes is not abolished when Psi is downregulated (Figure 3C,E,G). This could imply that an as yet unknown factor specifically promotes or represses tim splicing events in a temperature-dependent manner. Another possibility is that the strength of splice sites or tim’s pre-mRNA structure impacts splicing efficiency in a temperature–dependent manner. For example, suboptimal per splicing signals explain the lower efficiency of per’s most 3’ splicing event at warm temperature (Low et al., 2008).

How would the patterns of tim splicing affect the pace of the circadian clock, or advance the phase of circadian behavior under temperature cycles? In all splicing events that we studied, intron retention results in a truncated TIM protein. It is therefore possible that the balance of full length and truncated TIM proteins, which may function as endogenous dominant-negatives, determines circadian period. For example, truncated TIM might be less efficient at protecting PER from degradation, thus accelerating the pacemaker, or affecting its phase. Consistent with this idea, overexpression of the shorter cold-favored tim isoform (tim-sc) shortens period (Martin Anduaga et al., 2019). Strikingly, Psi downregulation increases this isoform’s levels and also results in a short phenotype. Shakhmantsir et al. (2018) also proposed that production of tim-M transcripts (called tim-tiny in their study) delays the rate of TIM accumulation. Such a mechanism could also contribute to the short period we observed when Psi is downregulated, since this reduces tim-M levels, which may accelerate TIM accumulation. Another interesting question is how PSI differentially affects specific splice isoforms of tim. One possibility is that the execution of a specific tim splicing event negatively influences the probability of the occurrence of other splicing events. For example, PSI could downregulate tim-sc and tim-cold by enhancing splicing and removal of the introns whose retention is necessary for production of these isoforms. This could indirectly reduce splicing of the intron that is retained in the warm tim-M isoform and result in tim-M upregulation. Conversely, PSI could directly promote tim-M intron retention and indirectly downregulate production of tim-sc and tim-cold.

Other splicing factors have been shown to be involved in the control of circadian rhythms in Drosophila. SRm160 contributes to the amplitude of circadian rhythms by promoting per expression (Beckwith et al., 2017), while B52/SMp55 and PRMT5 regulate per’s most 3’ splicing, which is temperature sensitive (Zhang et al., 2018; Sanchez et al., 2010). Loss of PRMT5 results in essentially arrhythmic behavior (Sanchez et al., 2010), but this is unlikely to be explained by its effect on per’s thermosensitive splicing. B52/SMp55 knockdown flies show a reduced siesta, which is controlled by the same per splicing (Zhang et al., 2018). With the identification of Psi, we uncover a key regulator of tim alternative splicing pattern and show that this pattern determines circadian period length, while per alternative splicing regulates the timing and amplitude of the daytime siesta. Interestingly, a recent study identified PRP4 kinase and other members of tri-snRNP complexes as regulators of circadian rhythms (Shakhmantsir et al., 2018). Downregulation of prp4 caused excessive retention of the tim-M intron. PSI and PRP4 might thus have complementary functions in tim mRNA splicing regulation, working together to maintain the proper balance of tim isoform expression.

An unexpected finding is the role played by both PDF neurons and other circadian neurons in the short period phenotype observed with circadian locomotor rhythms when we knocked-down Psi. Indeed, it is quite clear from multiple studies that under constant darkness, the PDF-positive sLNvs dictate the pace of circadian behavior (Stoleru et al., 2005; Yao and Shafer, 2014). Why, in the case of Psi downregulation, do PDF negative neurons also play a role in period determination? The explanation might be that PSI alters the hierarchy between circadian neurons, promoting the role of PDF negative neurons. This could be achieved by weakening PDF/PDFR signaling, for example.

While we focused our work on PSI, several other interesting candidates were identified in our screen (Tables 1 and 2). We note the presence of a large number of splicing factors. This adds to the emerging notion that alternative splicing plays a critical role in the control of circadian rhythms. We have already mentioned above several per splicing regulators that can impact circadian behavior. In addition, a recent study demonstrated that specific classes of circadian neurons express specific alternative splicing variants, and that rhythmic alternative splicing is widespread in these neurons (Wang et al., 2018). Interestingly, in this study, the splicing regulator barc, which was identified in our screen and which has been shown to causes intron retention in specific mRNAs (Abramczuk et al., 2017), was found to be rhythmically expressed in LNds. Moreover, in mammals, alternative splicing appears to be very sensitive to temperature, and could explain how body temperature rhythms synchronize peripheral clocks (Preußner et al., 2017). Another intriguing candidate is cg42458, which was found to be enriched in circadian neurons (LNvs and Dorsal Neurons 1) (Wang et al., 2018). In addition to emphasizing the role of splicing, our screen suggests that regulation of polyA tail length is important for circadian rhythmicity, since we identified several members of the CCR4-NOT complex and deadenylation-dependent decapping enzymes. Future work will be required to determine whether these factors directly target mRNAs encoding for core clock components, or whether their effect on circadian period is indirect. Interestingly, the POP2 deadenylase, which is part of the CCR4-NOT complex, was recently shown to regulate tim mRNA levels post-transcriptionally (Grima et al., 2019). It should be noted that while our screen targeted 364 proteins binding or associated with RNA, it did not include all of them. For example, LSM12, which was recently shown to be a part of the ATXN2/TYF complex (Lee et al., 2017), was not included in our screen because it had not been annotated as a potential RAP when we initiated our screen.

In summary, our work provides an important resource for identifying RNA associated proteins regulating circadian rhythms in Drosophila. It identifies PSI is an important regulator of circadian period and circadian phase in response to thermal cycles, and points at additional candidates and processes that determine the periodicity of circadian rhythms.

All source data are included in this submission.

1. 1. Wang Q
   2. Tallatero M
   3. Rio D

   (2016) NCBI Gene Expression Omnibus

   ID GSE79916. The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila.

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79916

1. 1. Abramczuk MK
   2. Burkard TR
   3. Rolland V
   4. Steinmann V
   5. Duchek P
   6. Jiang Y
   7. Wissel S
   8. Reichert H
   9. Knoblich JA

   (2017) The splicing co-factor barricade/Tat-SF1 is required for cell cycle and lineage progression in Drosophila neural stem cells

   Development 144:3932–3945.

   https://doi.org/10.1242/dev.152199

   • PubMed
   • Google Scholar
2. 1. Beckwith EJ
   2. Hernando CE
   3. Polcowñuk S
   4. Bertolin AP
   5. Mancini E
   6. Ceriani MF
   7. Yanovsky MJ

   (2017) Rhythmic behavior is controlled by the SRm160 splicing factor in Drosophila melanogaster

   Genetics 207:genetics.300139.2017.

   https://doi.org/10.1534/genetics.117.300139

   • PubMed
   • Google Scholar
3. 1. Boothroyd CE
   2. Wijnen H
   3. Naef F
   4. Saez L
   5. Young MW

   (2007) Integration of light and temperature in the regulation of circadian gene expression in Drosophila

   PLOS Genetics 3:e54.

   https://doi.org/10.1371/journal.pgen.0030054

   • PubMed
   • Google Scholar
4. 1. Bradley S
   2. Narayanan S
   3. Rosbash M

   (2012) NAT1/DAP5/p97 and atypical translational control in the Drosophila circadian oscillator

   Genetics 192:943–957.

   https://doi.org/10.1534/genetics.112.143248

   • PubMed
   • Google Scholar
5. 1. Busza A
   2. Emery-Le M
   3. Rosbash M
   4. Emery P

   (2004) Roles of the two Drosophila CRYPTOCHROME structural domains in circadian photoreception

   Science 304:1503–1506.

   https://doi.org/10.1126/science.1096973

   • PubMed
   • Google Scholar
6. 1. Busza A
   2. Murad A
   3. Emery P

   (2007) Interactions between circadian neurons control temperature synchronization of Drosophila behavior

   Journal of Neuroscience 27:10722–10733.

   https://doi.org/10.1523/JNEUROSCI.2479-07.2007

   • PubMed
   • Google Scholar
7. 1. Cao W
   2. Edery I

   (2017) Mid-day siesta in natural populations of D. Melanogaster from africa exhibits an altitudinal Cline and is regulated by splicing of a thermosensitive intron in the period clock gene

   BMC Evolutionary Biology 17:32.

   https://doi.org/10.1186/s12862-017-0880-8

   • PubMed
   • Google Scholar
8. 1. Cook KB
   2. Kazan H
   3. Zuberi K
   4. Morris Q
   5. Hughes TR

   (2011) RBPDB: a database of RNA-binding specificities

   Nucleic Acids Research 39:D301–D308.

   https://doi.org/10.1093/nar/gkq1069

   • PubMed
   • Google Scholar
9. 1. Dietzl G
   2. Chen D
   3. Schnorrer F
   4. Su KC
   5. Barinova Y
   6. Fellner M
   7. Gasser B
   8. Kinsey K
   9. Oppel S
   10. Scheiblauer S
   11. Couto A
   12. Marra V
   13. Keleman K
   14. Dickson BJ

   (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila

   Nature 448:151–156.

   https://doi.org/10.1038/nature05954

   • PubMed
   • Google Scholar
10. 1. Dubruille R
    2. Murad A
    3. Rosbash M
    4. Emery P

    (2009) A constant light-genetic screen identifies KISMET as a regulator of circadian photoresponses

    PLOS Genetics 5:e1000787.

    https://doi.org/10.1371/journal.pgen.1000787

    • PubMed
    • Google Scholar
11. 1. Duffy JB

    (2002) GAL4 system in Drosophila: a fly geneticist's Swiss army knife

    Genesis 34:1–15.

    https://doi.org/10.1002/gene.10150

    • PubMed
    • Google Scholar
12. 1. Dunlap JC

    (1999) Molecular bases for circadian clocks

    Cell 96:271–290.

    https://doi.org/10.1016/S0092-8674(00)80566-8

    • PubMed
    • Google Scholar
13. 1. Emery P
    2. So WV
    3. Kaneko M
    4. Hall JC
    5. Rosbash M

    (1998) CRY, a Drosophila clock and light-regulated cryptochrome, is a major contributor to circadian rhythm resetting and photosensitivity

    Cell 95:669–679.

    https://doi.org/10.1016/S0092-8674(00)81637-2

    • PubMed
    • Google Scholar
14. 1. Green EW
    2. Fedele G
    3. Giorgini F
    4. Kyriacou CP

    (2014) A Drosophila RNAi collection is subject to dominant phenotypic effects

    Nature Methods 11:222–223.

    https://doi.org/10.1038/nmeth.2856

    • PubMed
    • Google Scholar
15. 1. Grima B
    2. Lamouroux A
    3. Chélot E
    4. Papin C
    5. Limbourg-Bouchon B
    6. Rouyer F

    (2002) The F-box protein slimb controls the levels of clock proteins period and timeless

    Nature 420:178–182.

    https://doi.org/10.1038/nature01122

    • PubMed
    • Google Scholar
16. 1. Grima B
    2. Papin C
    3. Martin B
    4. Chélot E
    5. Ponien P
    6. Jacquet E
    7. Rouyer F

    (2019) PERIOD-controlled deadenylation of the timeless transcript in the Drosophila circadian clock

    PNAS 116:5721–5726.

    https://doi.org/10.1073/pnas.1814418116

    • PubMed
    • Google Scholar
17. 1. Guo L
    2. Zaysteva O
    3. Nie Z
    4. Mitchell NC
    5. Amanda Lee JE
    6. Ware T
    7. Parsons L
    8. Luwor R
    9. Poortinga G
    10. Hannan RD
    11. Levens DL
    12. Quinn LM

    (2016) Defining the essential function of FBP/KSRP proteins: Drosophila psi interacts with the mediator complex to modulate MYC transcription and tissue growth

    Nucleic Acids Research 44:7646–7658.

    https://doi.org/10.1093/nar/gkw461

    • PubMed
    • Google Scholar
18. 1. Hardin PE

    (2011) Molecular genetic analysis of circadian timekeeping in Drosophila

    Advances in Genetics 74:141–173.

    https://doi.org/10.1016/B978-0-12-387690-4.00005-2

    • PubMed
    • Google Scholar
19. 1. Harrisingh MC
    2. Wu Y
    3. Lnenicka GA
    4. Nitabach MN

    (2007) Intracellular ^Ca2+regulates free-running circadian clock oscillation in vivo

    Journal of Neuroscience 27:12489–12499.

    https://doi.org/10.1523/JNEUROSCI.3680-07.2007

    • PubMed
    • Google Scholar
20. 1. Huang Y
    2. McNeil GP
    3. Jackson FR

    (2014) Translational regulation of the DOUBLETIME/CKIδ/ε kinase by LARK contributes to circadian period modulation

    PLOS Genetics 10:e1004536.

    https://doi.org/10.1371/journal.pgen.1004536

    • PubMed
    • Google Scholar
21. 1. Kadener S
    2. Stoleru D
    3. McDonald M
    4. Nawathean P
    5. Rosbash M

    (2007) Clockwork orange is a transcriptional repressor and a new Drosophila circadian pacemaker component

    Genes & Development 21:1675–1686.

    https://doi.org/10.1101/gad.1552607

    • PubMed
    • Google Scholar
22. 1. Kaneko M
    2. Park JH
    3. Cheng Y
    4. Hardin PE
    5. Hall JC

    (2000) Disruption of synaptic transmission or clock-gene-product oscillations in circadian pacemaker cells of Drosophila cause abnormal behavioral rhythms

    Journal of Neurobiology 43:207–233.

    https://doi.org/10.1002/(SICI)1097-4695(20000605)43:3<207::AID-NEU1>3.0.CO;2-0

    • PubMed
    • Google Scholar
23. 1. Koh K
    2. Zheng X
    3. Sehgal A

    (2006) JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS

    Science 312:1809–1812.

    https://doi.org/10.1126/science.1124951

    • PubMed
    • Google Scholar
24. 1. Kojima S
    2. Matsumoto K
    3. Hirose M
    4. Shimada M
    5. Nagano M
    6. Shigeyoshi Y
    7. Hoshino S
    8. Ui-Tei K
    9. Saigo K
    10. Green CB
    11. Sakaki Y
    12. Tei H

    (2007) LARK activates posttranscriptional expression of an essential mammalian clock protein, PERIOD1

    PNAS 104:1859–1864.

    https://doi.org/10.1073/pnas.0607567104

    • PubMed
    • Google Scholar
25. 1. Labourier E
    2. Adams MD
    3. Rio DC

    (2001) Modulation of P-element pre-mRNA splicing by a direct interaction between PSI and U1 snRNP 70K protein

    Molecular Cell 8:363–373.

    https://doi.org/10.1016/S1097-2765(01)00311-2

    • PubMed
    • Google Scholar
26. 1. Labourier E
    2. Blanchette M
    3. Feiger JW
    4. Adams MD
    5. Rio DC

    (2002) The KH-type RNA-binding protein PSI is required for Drosophila viability, male fertility, and cellular mRNA processing

    Genes & Development 16:72–84.

    https://doi.org/10.1101/gad.948602

    • PubMed
    • Google Scholar
27. 1. Lamba P
    2. Foley LE
    3. Emery P

    (2018) Neural network interactions modulate CRY-Dependent photoresponses in Drosophila

    The Journal of Neuroscience 38:6161–6171.

    https://doi.org/10.1523/JNEUROSCI.2259-17.2018

    • PubMed
    • Google Scholar
28. 1. Lee J
    2. Yoo E
    3. Lee H
    4. Park K
    5. Hur JH
    6. Lim C

    (2017) LSM12 and ME31B/DDX6 define distinct modes of posttranscriptional regulation by ATAXIN-2 protein complex in Drosophila circadian pacemaker neurons

    Molecular Cell 66:129–140.

    https://doi.org/10.1016/j.molcel.2017.03.004

    • PubMed
    • Google Scholar
29. 1. Levine JD
    2. Funes P
    3. Dowse HB
    4. Hall JC

    (2002) Signal analysis of behavioral and molecular cycles

    BMC Neuroscience 3:5.

    https://doi.org/10.1186/1471-2202-3-1

    • Google Scholar
30. 1. Lim C
    2. Chung BY
    3. Pitman JL
    4. McGill JJ
    5. Pradhan S
    6. Lee J
    7. Keegan KP
    8. Choe J
    9. Allada R

    (2007) Clockwork orange encodes a transcriptional repressor important for circadian-clock amplitude in Drosophila

    Current Biology 17:1082–1089.

    https://doi.org/10.1016/j.cub.2007.05.039

    • PubMed
    • Google Scholar
31. 1. Lim C
    2. Lee J
    3. Choi C
    4. Kilman VL
    5. Kim J
    6. Park SM
    7. Jang SK
    8. Allada R
    9. Choe J

    (2011) The novel gene twenty-four defines a critical translational step in the Drosophila clock

    Nature 470:399–403.

    https://doi.org/10.1038/nature09728

    • PubMed
    • Google Scholar
32. 1. Lim C
    2. Allada R

    (2013a) ATAXIN-2 activates PERIOD translation to sustain circadian rhythms in Drosophila

    Science 340:875–879.

    https://doi.org/10.1126/science.1234785

    • PubMed
    • Google Scholar
33. 1. Lim C
    2. Allada R

    (2013b) Emerging roles for post-transcriptional regulation in circadian clocks

    Nature Neuroscience 16:1544–1550.

    https://doi.org/10.1038/nn.3543

    • PubMed
    • Google Scholar
34. 1. Lin FJ
    2. Song W
    3. Meyer-Bernstein E
    4. Naidoo N
    5. Sehgal A

    (2001) Photic signaling by cryptochrome in the Drosophila circadian system

    Molecular and Cellular Biology 21:7287–7294.

    https://doi.org/10.1128/MCB.21.21.7287-7294.2001

    • PubMed
    • Google Scholar
35. 1. Low KH
    2. Lim C
    3. Ko HW
    4. Edery I

    (2008) Natural variation in the splice site strength of a clock gene and species-specific thermal adaptation

    Neuron 60:1054–1067.

    https://doi.org/10.1016/j.neuron.2008.10.048

    • PubMed
    • Google Scholar
36. 1. Low KH
    2. Chen WF
    3. Yildirim E
    4. Edery I

    (2012) Natural variation in the Drosophila Melanogaster clock gene period modulates splicing of its 3'-terminal intron and mid-day siesta

    PLOS ONE 7:e49536.

    https://doi.org/10.1371/journal.pone.0049536

    • PubMed
    • Google Scholar
37. 1. Majercak J
    2. Sidote D
    3. Hardin PE
    4. Edery I

    (1999) How a circadian clock adapts to seasonal decreases in temperature and day length

    Neuron 24:219–230.

    https://doi.org/10.1016/S0896-6273(00)80834-X

    • PubMed
    • Google Scholar
38. 1. Majercak J
    2. Chen WF
    3. Edery I

    (2004) Splicing of the period gene 3'-terminal intron is regulated by light, circadian clock factors, and phospholipase C

    Molecular and Cellular Biology 24:3359–3372.

    https://doi.org/10.1128/MCB.24.8.3359-3372.2004

    • PubMed
    • Google Scholar
39. 1. Martin Anduaga A
    2. Evantal N
    3. Patop IL
    4. Bartok O
    5. Weiss R
    6. Kadener S

    (2019) Thermosensitive alternative splicing senses and mediates temperature adaptation in Drosophila

    eLife 8:e44642.

    https://doi.org/10.7554/eLife.44642

    • PubMed
    • Google Scholar
40. 1. Matsumoto A
    2. Ukai-Tadenuma M
    3. Yamada RG
    4. Houl J
    5. Uno KD
    6. Kasukawa T
    7. Dauwalder B
    8. Itoh TQ
    9. Takahashi K
    10. Ueda R
    11. Hardin PE
    12. Tanimura T
    13. Ueda HR

    (2007) A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock

    Genes & Development 21:1687–1700.

    https://doi.org/10.1101/gad.1552207

    • PubMed
    • Google Scholar
41. 1. Myers MP
    2. Wager-Smith K
    3. Wesley CS
    4. Young MW
    5. Sehgal A

    (1995) Positional cloning and sequence analysis of the Drosophila clock gene, timeless

    Science 270:805–808.

    https://doi.org/10.1126/science.270.5237.805

    • PubMed
    • Google Scholar
42. 1. Pittendrigh CS

    (1960) Circadian rhythms and the circadian organization of living systems

    Cold Spring Harbor Symposia on Quantitative Biology 25:159–184.

    https://doi.org/10.1101/SQB.1960.025.01.015

    • PubMed
    • Google Scholar
43. 1. Preußner M
    2. Goldammer G
    3. Neumann A
    4. Haltenhof T
    5. Rautenstrauch P
    6. Müller-McNicoll M
    7. Heyd F

    (2017) Body temperature cycles control rhythmic alternative splicing in mammals

    Molecular Cell 67:433–446.

    https://doi.org/10.1016/j.molcel.2017.06.006

    • PubMed
    • Google Scholar
44. 1. Renn SC
    2. Park JH
    3. Rosbash M
    4. Hall JC
    5. Taghert PH

    (1999) A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila

    Cell 99:791–802.

    https://doi.org/10.1016/S0092-8674(00)81676-1

    • PubMed
    • Google Scholar
45. 1. Ri H
    2. Lee J
    3. Sonn JY
    4. Yoo E
    5. Lim C
    6. Choe J

    (2019) Drosophila CrebB is a substrate of the Nonsense-Mediated mRNA decay pathway that sustains circadian behaviors

    Molecules and Cells 42:301–312.

    https://doi.org/10.14348/molcells.2019.2451

    • PubMed
    • Google Scholar
46. 1. Richier B
    2. Michard-Vanhée C
    3. Lamouroux A
    4. Papin C
    5. Rouyer F

    (2008) The clockwork orange Drosophila protein functions as both an activator and a repressor of clock gene expression

    Journal of Biological Rhythms 23:103–116.

    https://doi.org/10.1177/0748730407313817

    • PubMed
    • Google Scholar
47. 1. Rutila JE
    2. Maltseva O
    3. Rosbash M

    (1998) The timSL mutant affects a restricted portion of the Drosophila Melanogaster circadian cycle

    Journal of Biological Rhythms 13:380–392.

    https://doi.org/10.1177/074873098129000200

    • PubMed
    • Google Scholar
48. 1. Sanchez SE
    2. Petrillo E
    3. Beckwith EJ
    4. Zhang X
    5. Rugnone ML
    6. Hernando CE
    7. Cuevas JC
    8. Godoy Herz MA
    9. Depetris-Chauvin A
    10. Simpson CG
    11. Brown JW
    12. Cerdán PD
    13. Borevitz JO
    14. Mas P
    15. Ceriani MF
    16. Kornblihtt AR
    17. Yanovsky MJ

    (2010) A methyl transferase links the circadian clock to the regulation of alternative splicing

    Nature 468:112–116.

    https://doi.org/10.1038/nature09470

    • PubMed
    • Google Scholar
49. 1. Sehgal A
    2. Price JL
    3. Man B
    4. Young MW

    (1994) Loss of circadian behavioral rhythms and per RNA oscillations in the Drosophila mutant timeless

    Science 263:1603–1606.

    https://doi.org/10.1126/science.8128246

    • PubMed
    • Google Scholar
50. 1. Shakhmantsir I
    2. Nayak S
    3. Grant GR
    4. Sehgal A

    (2018) Spliceosome factors target timeless (tim) mRNA to control clock protein accumulation and circadian behavior in Drosophila

    eLife 7:e39821.

    https://doi.org/10.7554/eLife.39821

    • PubMed
    • Google Scholar
51. 1. Siebel CW
    2. Fresco LD
    3. Rio DC

    (1992) The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding

    Genes & Development 6:1386–1401.

    https://doi.org/10.1101/gad.6.8.1386

    • PubMed
    • Google Scholar
52. 1. So WV
    2. Rosbash M

    (1997) Post-transcriptional regulation contributes to Drosophila clock gene mRNA cycling

    The EMBO Journal 16:7146–7155.

    https://doi.org/10.1093/emboj/16.23.7146

    • PubMed
    • Google Scholar
53. 1. Stanewsky R
    2. Jamison CF
    3. Plautz JD
    4. Kay SA
    5. Hall JC

    (1997) Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila

    The EMBO Journal 16:5006–5018.

    https://doi.org/10.1093/emboj/16.16.5006

    • PubMed
    • Google Scholar
54. 1. Stanewsky R
    2. Kaneko M
    3. Emery P
    4. Beretta B
    5. Wager-Smith K
    6. Kay SA
    7. Rosbash M
    8. Hall JC

    (1998) The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila

    Cell 95:681–692.

    https://doi.org/10.1016/S0092-8674(00)81638-4

    • PubMed
    • Google Scholar
55. 1. Stoleru D
    2. Peng Y
    3. Agosto J
    4. Rosbash M

    (2004) Coupled oscillators control morning and evening locomotor behaviour of Drosophila

    Nature 431:862–868.

    https://doi.org/10.1038/nature02926

    • PubMed
    • Google Scholar
56. 1. Stoleru D
    2. Peng Y
    3. Nawathean P
    4. Rosbash M

    (2005) A resetting signal between Drosophila pacemakers synchronizes morning and evening activity

    Nature 438:238–242.

    https://doi.org/10.1038/nature04192

    • PubMed
    • Google Scholar
57. 1. Tataroglu O
    2. Emery P

    (2015) The molecular ticks of the Drosophila circadian clock

    Current Opinion in Insect Science 7:51–57.

    https://doi.org/10.1016/j.cois.2015.01.002

    • PubMed
    • Google Scholar
58. 1. Thurmond J
    2. Goodman JL
    3. Strelets VB
    4. Attrill H
    5. Gramates LS
    6. Marygold SJ
    7. Matthews BB
    8. Millburn G
    9. Antonazzo G
    10. Trovisco V
    11. Kaufman TC
    12. Calvi BR
    13. FlyBase Consortium

    (2019) FlyBase 2.0: the next generation

    Nucleic Acids Research 47:D759–D765.

    https://doi.org/10.1093/nar/gky1003

    • PubMed
    • Google Scholar
59. 1. Vissers JH
    2. Manning SA
    3. Kulkarni A
    4. Harvey KF

    (2016) A Drosophila RNAi library modulates hippo pathway-dependent tissue growth

    Nature Communications 7:10368.

    https://doi.org/10.1038/ncomms10368

    • PubMed
    • Google Scholar
60. 1. Wang Q
    2. Taliaferro JM
    3. Klibaite U
    4. Hilgers V
    5. Shaevitz JW
    6. Rio DC

    (2016) The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila

    PNAS 113:5269–5274.

    https://doi.org/10.1073/pnas.1600936113

    • PubMed
    • Google Scholar
61. 1. Wang Q
    2. Abruzzi KC
    3. Rosbash M
    4. Rio DC

    (2018) Striking circadian neuron diversity and cycling of Drosophila alternative splicing

    eLife 7:e35618.

    https://doi.org/10.7554/eLife.35618

    • PubMed
    • Google Scholar
62. 1. Yao Z
    2. Shafer OT

    (2014) The Drosophila circadian clock is a variably coupled network of multiple peptidergic units

    Science 343:1516–1520.

    https://doi.org/10.1126/science.1251285

    • PubMed
    • Google Scholar
63. 1. Zerbino DR
    2. Achuthan P
    3. Akanni W
    4. Amode MR
    5. Barrell D
    6. Bhai J
    7. Billis K
    8. Cummins C
    9. Gall A
    10. Girón CG
    11. Gil L
    12. Gordon L
    13. Haggerty L
    14. Haskell E
    15. Hourlier T
    16. Izuogu OG
    17. Janacek SH
    18. Juettemann T
    19. To JK
    20. Laird MR
    21. Lavidas I
    22. Liu Z
    23. Loveland JE
    24. Maurel T
    25. McLaren W
    26. Moore B
    27. Mudge J
    28. Murphy DN
    29. Newman V
    30. Nuhn M
    31. Ogeh D
    32. Ong CK
    33. Parker A
    34. Patricio M
    35. Riat HS
    36. Schuilenburg H
    37. Sheppard D
    38. Sparrow H
    39. Taylor K
    40. Thormann A
    41. Vullo A
    42. Walts B
    43. Zadissa A
    44. Frankish A
    45. Hunt SE
    46. Kostadima M
    47. Langridge N
    48. Martin FJ
    49. Muffato M
    50. Perry E
    51. Ruffier M
    52. Staines DM
    53. Trevanion SJ
    54. Aken BL
    55. Cunningham F
    56. Yates A
    57. Flicek P

    (2018) Ensembl 2018

    Nucleic Acids Research 46:D754–D761.

    https://doi.org/10.1093/nar/gkx1098

    • PubMed
    • Google Scholar
64. 1. Zhang Y
    2. Ling J
    3. Yuan C
    4. Dubruille R
    5. Emery P

    (2013) A role for Drosophila ATX2 in activation of PER translation and circadian behavior

    Science 340:879–882.

    https://doi.org/10.1126/science.1234746

    • PubMed
    • Google Scholar
65. 1. Zhang Z
    2. Cao W
    3. Edery I

    (2018) The SR protein B52/SRp55 regulates splicing of the period thermosensitive intron and mid-day siesta in Drosophila

    Scientific Reports 8:1872.

    https://doi.org/10.1038/s41598-017-18167-3

    • PubMed
    • Google Scholar
66. 1. Zhang Y
    2. Emery P

    (2013) GW182 controls Drosophila circadian behavior and PDF-receptor signaling

    Neuron 78:152–165.

    https://doi.org/10.1016/j.neuron.2013.01.035

    • PubMed
    • Google Scholar
67. 1. Zheng X
    2. Koh K
    3. Sowcik M
    4. Smith CJ
    5. Chen D
    6. Wu MN
    7. Sehgal A

    (2009) An isoform-specific mutant reveals a role of PDP1 epsilon in the circadian oscillator

    Journal of Neuroscience 29:10920–10927.

    https://doi.org/10.1523/JNEUROSCI.2133-09.2009

    • PubMed
    • Google Scholar

Acceptance summary:

A transcriptional feedback loop comprising of PER/TIMELESS and CLOCK/CYCLE underlies periodic oscillation of the circadian clock and diurnal rhythms. However, the activity of these core components is modulated by post-transcriptional and post-translational processes that remain to be fully understood. This work, by Foley and coauthors, begins to address post-transcriptional control mechanisms by screening Drosophila genes encoding RNA-binding and RNA-associated proteins for their role in the control of circadian rhythms. From 364 genes screened a total of 43 candidates (12%) appear to alter period length. The unexpectedly high hit rate suggests that RNA regulation plays a significant role in clock function. The authors then focus on one of these proteins, PSI, and reveal that it encodes a splicing factor that produces timeless splice isoforms described and characterised in a companion paper by Anduaga et al. in this issue of eLife. Several observations support a model in which PSI is required to mediate alternative splicing of tim isoforms, which in turn determines how the phase of the circadian cycles adapts in response to temperature variation. Observations made here suggest that PSI is required for appropriate tim mRNA splicing in both cold and warm conditions, and acts downstream of a primary temperature-sensing mechanism. Additional work is needed to identify how exactly temperature-specific splicing patterns of TIM are determined.

Decision letter after peer review:

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "PSI controls tim splicing and

circadian period in Drosophila" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Mani Ramaswami as Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

The reviewers concur that your careful screen of RNA-binding proteins is well done and broadly useful. However, the consensus view is that the behavioural evidence to support the model that PSI mediated splicing of Tim contributes to locomotor rhythms is too subtle to be convincing. It also remains possible that thermal adaption affected by psi mutations have nothing to do with locomotor rhythms but perhaps to with do other non-rhythmic phenotypes. Thus, the feeling is that despite the interest of the subject and the overall quality of the work, the manuscript falls short of biological insight required for acceptance.

Reviewer #1:

This is an interesting paper focusing on a splicing factor PSI that regulates timeless isoforms. The screening and experimental work is done well with care taken to exclude background effects. One of the disappointing aspects of the study, and this is no fault of the authors, is the lack of any striking disruption of temperature adaptations with the PSI KD. With all these changes in timeless and cwo splicing, particularly of the temperature sensitive tim isoforms, the locomotor rhythms of the psi KD flies in LD cycles is normal without the kind of dramatic effects seen on siestas when per 3' splicing is disturbed. So I was left scratching my head as to what disruption of psi does phenotypically – small changes in period, sure, but these are not reflected in the LD profiles of Figure 3—figure supplement 2.

As the period seems to change with psi manipulation, I was surprised that the authors did not perform a simple temperature compensation experiment (or did I miss it?). I realize that gal4 is temperature sensitive but the direction of any period change between 18-30° C might have been interesting. Are there compensatory changes of tim transcripts in psi KD? The accompanying paper hinted at that with the cold-sensitive isoforms.

The loss of rhythmicity in Figure 2E and 2G on psi overexpression is striking. Any idea what happens to tim isoforms under these conditions? Clearly something in the dorsal neurons is having a major effect on the rhythmicity of PDF neurons. This is interesting as the DNs do not by themselves generate locomotor rhythms. Surprisingly, the Discussion does not mention this most dramatic result of the manuscript.

Reviewer #2:

This is a very interesting paper from the Kadener and Emery labs about a genetic screen for RNA-binding and RNA-associated genes involved in circadian clock control. After screening 364 genes, they found a total of 43 candidates (12%) that alter period length. Compared to standard genetic screens, this is a very high hit rate, indicating that RNA regulation plays a significant role in clock regulation. If correct, this is an important discovery revealing yet another regulatory level of the already very complex circadian clock mechanism.

The paper is well written and experiments conducted are of high quality and according to the standard in the field. The authors focus on one gene identified in their screen (psi), which they show causes period shortening when down-regulated and arrhythmicity when overexpressed in the entire clock circuit. Effects with more narrow down regulation (Pdf-gal4) are much less pronounced and overall it remains unclear in which parts of the circuit Psi plays a role. Moreover important controls are lacking in the behavior experiments (outlined below). Because the effects on period are rather mild, these controls are important. Also in light of the fact that psi has no measurable effect on the light PRC or normal LD entrainment it seems important to really nail the effect on period. Molecularly it seems clear that psi affects tim-splicing, but the mechanism remains elusive and it is not clear how psi can promote intron retention in certain tim transcripts at cooler temperature and that in other tim transcripts at warmer temperatures.

1) The period shortenings after Psi-knockdown are relatively mild (0.2-1.2 hr depending on the driver used) and the same applies for the lengthening observed after psi-overexpression (0.1 – 0.7 hr) (Figure 2 and Table 3). Given that these period changes are key to the message of the paper (psi controls circadian period), I think it is important to perform a statistical analysis comparing the various genotypes. Also, some key controls are missing: the psi RNAi lines have not been tested without a driver. Data are shown for the total of RNAi-lines from a given collection over + in Figure 1A, but due to the large variability between the different collections with regard to period length, the values for the individual RNAi lines 'over +' must be given. I also checked the source file (Dataset 1) and found some data, which I think shows the RNAi/+ data. From these data it looks the effect of the psi knockdown is even smaller (psiRNAi-kk/+: 24.0, TD2>kk: 23.6, PD2>kk:24.7 and psiRNAi-GD/+ 24.2, TD2>GD: 23.8, PD2>GD: 24.2). So no effect with the Pdf driver on period at all, and a small 0.4 hr effect with TD2. Moreover, no effects with 2 other RNAi lines shown in this Data set with either tim or Pdf drivers. I am not sure if the authors have more control data and if the Data set table represents only the actual screening data, but clearly additional controls are required. This is an important point, because no other behavioral phenotypes after psi knockdown could be observed (LD behavior at different temperatures and light-PRC, Figure 3—figure supplements 2 and 3).

2) The authors see stronger effects with tim-gal4 (both knock-down and overexpression of psi) compared to Pdf-gal4. They aim to distinguish between potentially stronger tim-gal4 expression in the s-LNv, or a period-determining function of non-s-LNv neurons by applying a tim-gal4/Pdf-gal80 combination, which should eliminate gal4 from the s-LNv but not all the other clock cells. Unfortunately, this experiment did not allow for a clear distinction, as the period shortening after knockdown was in between that of tim and Pdf drivers alone. Overexpression with the tim-gal4/Pdf-gal80 combination recapitulated the high percentage of arhythmicity seen with tim alone, but not the period-lengthening, whereas overexpression with Pdf-gal4 only had no effect. Overall the results suggest a role for non-s-LNv neurons in setting period length and controlling rhythmicity, but I think this should be repeated with other, non-s-LNv drivers to get a clearer picture (e.g., it cannot be ruled out that Pdf-gal80 is completely blocking gal4 in the s-LNv). I suggest using the splitE cell-gal4 and drivers specific for the DN1 to solve this issue.

3) psi overexpression with tim-gal4 and tim-gal4/Pdf-gal80 produces very high levels of arrhythmicity and the few rhythmic flies (n=5 and n=4, respectively) have low average power values. Considering the low number of rhythmic flies and the low power values, I find it problematical to claim that overexpression with tim-gal4 causes period-lengthening. Could the authors show actograms that clearly show the longer period in these flies compared to the tim-gal4/Pdf-gal80 flies. Lacking additional support (such as convincing actograms and/or higher n's), I think it is not OK to conclude that the overexpression results are in line with the knockdown results (which are problematical in itself, see major point 1 above).

4) The peripheral clock data in Figure 3 do not look very convincing, due to the poor rhythmicity of the luciferase oscillations in DD. As correctly mentioned in the Materials and methods part, it is expected and was reported previously that rhythms of these reporters dampen rapidly in DD. But I am not sure if it is valid to calculate period values from 48 hr only, particularly if one the 2nd day the oscillations are very low amplitude. Perhaps it would be better to look in LD (where the reporters cycle with high-amplitude) to see if the rhythms in psi knock down flies are slightly phase-advanced? Or use dissected peripheral tissues in hope of stronger rhythms in DD (whole body rhythms could be further dampened due to internal desynchronization between tissues).

5) Figure 5 and accompanying Results text. I think this is the key figure about the molecular effects of reducing psi function (the effects on cwo are relatively weak and much less convincing). The data in Figure 5 clearly show that tim introns that are usually retained at cold temperatures (due to no splicing at the usual splice sites, I assume) and lead to higher mRNA levels of these particular transcripts, are also elevated at 25°C when psi is being knocked down. So it seems that psi is responsible for splicing out these introns at warmer temperatures. In contrast, an intron normally retained at high temperatures (29°C) and resulting in high tim-M transcript levels is spliced out in psi knockdown heads at 25°C, leading to lower tim-M levels both at 25°C and 29°C. This suggests that for this transcript, psi seems to promote intron retention (so to reduce splicing) at warmer temperatures. How can this be explained by a common molecular mechanism? Also, it doesn't help that the authors refer to an accompanying paper (not available for this reviewer) for the nature of the different tim transcripts and splicing events. Without a map explaining these mainly totally novel alternative tim transcripts it is impossible to follow what is going on. So, a map seems mandatory, also because the reader should not be forced to swap to a different paper in order to understand the current one.

6) Figure 6 supports the idea that psi regulates period by tim splicing. I am bit confused by the genotype description used in the figure and Table 3. In the other parts of the paper tim-gal4 UAS-dicer2 was abbreviated as TD2, but here 'TG4/+;UAS-Dcr2/+ was used. Are these the same flies or constructs on different chromosomes. Please explain. Also, as in Figure 2, the controls for RNAi/+ are missing.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "Drosophila PSI controls circadian period and the phase of circadian behavior under temperature cycle via tim splicing" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Mani Ramaswami as Reviewing Editor and Ronald Calabrese as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This is a resubmitted version of a previously declined article that has been substantially improved. The work described begins with a genetic screen for RNA-binding and RNA-associated genes involved in circadian clock control. Of 364 genes screened, a total of 43 candidates (12%) appear to alter period length. The unexpectedly high hit rate compared to standard genetic screens is significant and interesting, suggesting that RNA regulation plays a significant role in clock regulation. The authors go on to focus on one of these, PSI, a splicing factor that regulates timeless isoforms. Various observations support a model in which PSI controlled temperature-dependent-splicing regulates the circadian period and determines how the phase of the circadian cycles adapts in response to temperature cycles. However, there remain several ambiguities, missing data, controls and issues that need to be addressed.

Essential revisions:

1) Figure 2C to H. At 30°C there are clear period-shortening effects (panel 2C). But from Table 3 and it's difficult for the reader to extract from these numbers whether the UAS-RNAi, and UAS-Psi constructs are significantly different from the experimentals at 25°C. Where are the UAS-Psi RNAi and UAS-Psi overexpression controls for 25°C work? Without these it's difficult to assess how valid are the period changes at 25°C. These should be included. Some of the statements being presented from Figure 2 are possibly incorrect e.g. panel 2D. The authors need to present the 25°C UAS/+ results graphically in Figure 2 and perform the appropriate statistical comparisons. In fact the UAS psi/+ result would actually strengthen their case for period lengthening for panels F and H.

2) Figure 3A and B isn't so convincing visually because there is only one cycle, even though the cosinor method used is appropriate. Might this be relegated to a supplementary figure as it adds very little to the manuscript?

3) Further in Figure 3: It is still very difficult to see the period shortening in the bioluminescence traces (B, D). In particular it is difficult to distinguish between the black circles and black squares (RNAi/+ and test flies, respectively). It would help to at least use colored symbols to help distinguishing the different genotypes.

4) Figure 4 does not add much to this manuscript, where two cwo splice forms appear to be reduced and one is increased. The results are described briefly then immediately dropped – this could be relegated to a supplementary figure.

5) The tim splicing results are very confusing and need to be clarified. The cold tim isoforms (tim-cold and tim-sc) are elevated at warm temperatures in the psi mutant so normally psi would enhance splicing at cold temperatures. The warmer tim isoform (tim-M) is reduced at 25°C in the psi mutant so we'd presume that psi normally enhances this isoform at warm temperatures. Therefore psi normally has opposite effects on the tim isoforms, so a general temperature-sensitive psi mechanism appears to be excluded.

6) Figure 4—figure supplement 1 the UAS RNAi controls are not shown here. Are they temperature compensated? The Td2 control is not temperature compensated and has a relatively large change in period at hot temperature.

7) 'Collectively, these results indicate that PSI shifts the balance toward a warm temperature tim RNA isoform profile at an intermediate temperature (25°C).' This sentence makes no sense – no general mechanism can emerge as PSI appears to have opposite effects on the cold/warm tim isoforms. It enhances the warm forms and reduces the cold forms. Do the authors mean wild-type PSI or the psi-mutant? Please clarify.

[Editors’ note: the author responses to the first round of peer review follow.]

We would like to thank the two reviewers for their insightful comments. We believe that by addressing them with additional experiments and improved discussion, we have very significantly improved our manuscript. The most important additions are the followings. First, we have discovered that PSI downregulation advances the phase of circadian behavior under temperature cycles. This phenotype is remarkably strong and reveal a specific role for PSI in circadian thermal response. Second, we have also significantly strengthened the data supporting a role for PSI in period control.

Reviewer #1:

This is an interesting paper focusing on a splicing factor PSI that regulates timeless isoforms. The screening and experimental work is done well with care taken to exclude background effects. One of the disappointing aspects of the study, and this is no fault of the authors, is the lack of any striking disruption of temperature adaptations with the PSI KD. With all these changes in timeless and cwo splicing, particularly of the temperature sensitive tim isoforms, the locomotor rhythms of the psi KD flies in LD cycles is normal without the kind of dramatic effects seen on siestas when per 3' splicing is disturbed. So I was left scratching my head as to what disruption of psi does phenotypically – small changes in period, sure, but these are not reflected in the LD profiles of Figure 3—figure supplement 2.

As the period seems to change with psi manipulation, I was surprised that the authors did not perform a simple temperature compensation experiment (or did I miss it?). I realize that gal4 is temperature sensitive but the direction of any period change between 18-30°C might have been interesting. Are there compensatory changes of tim transcripts in psi KD? The accompanying paper hinted at that with the cold-sensitive isoforms.

We thank the reviewer for his interest in our work. We did perform a temperature compensation experiment but did not observe a phenotype. We have added these results to the manuscript. However, we have now also performed temperature entrainment experiments and observed a striking phenotype.The phase of circadian behavior is advanced by several hours under a temperature cycle. It is however not advanced under a light/dark cycle. Moreover, in flies that cannot splice tim in a temperature dependent manner, the phase of circadian behavior is not advanced. Therefore, PSI specifically regulates the phase of circadian behavior in the presence of a temperature cycle, through regulation of tim splicing.

The loss of rhythmicity in Figure 2E and 2G on psi overexpression is striking. Any idea what happens to tim isoforms under these conditions? Clearly something in the dorsal neurons is having a major effect on the rhythmicity of PDF neurons. This is interesting as the DNs do not by themselves generate locomotor rhythms. Surprisingly, the Discussion does not mention this most dramatic result of the manuscript.

Reviewer 2 also showed interest in this phenotype, which we tried to attribute to a specific group of circadian neurons. Unfortunately, the results are not straightforward, and suggest that arrhythmicity is the sum of PSI downregulation in multiple groups of circadian neurons. Also, as we discussed in the original manuscript, arrhythmicity is more difficult to interpret than a period phenotype. We would thus prefer not to include these data in the present manuscript, particularly since we have now identified a striking temperature-dependent phenotype. However, if the editors and reviewers concur that we should add these data, we will provide them.

Reviewer #2:

[…] The paper is well written and experiments conducted are of high quality and according to the standard in the field. The authors focus on one gene identified in their screen (psi), which they show causes period shortening when down-regulated and arrhythmicity when overexpressed in the entire clock circuit. Effects with more narrow down regulation (Pdf-gal4) are much less pronounced and overall it remains unclear in which parts of the circuit Psi plays a role. Moreover important controls are lacking in the behavior experiments (outlined below). Because the effects on period are rather mild, these controls are important. Also in light of the fact that psi has no measurable effect on the light PRC or normal LD entrainment it seems important to really nail the effect on period. Molecularly it seems clear that psi affects tim-splicing, but the mechanism remains elusive and it is not clear how psi can promote intron retention in certain tim transcripts at cooler temperature and that in other tim transcripts at warmer temperatures.

1) The period shortenings after Psi-knockdown are relatively mild (0.2-1.2 hr depending on the driver used) and the same applies for the lengthening observed after psi-overexpression (0.1 – 0.7 hr) (Figure 2 and Table 3). Given that these period changes are key to the message of the paper (psi controls circadian period), I think it is important to perform a statistical analysis comparing the various genotypes. Also, some key controls are missing: the psi RNAi lines have not been tested without a driver. Data are shown for the total of RNAi-lines from a given collection over + in Figure 1A, but due to the large variability between the different collections with regard to period length, the values for the individual RNAi lines 'over +' must be given. I also checked the source file (Dataset 1) and found some data, which I think shows the RNAi/+ data. From these data it looks the effect of the psi knockdown is even smaller (psiRNAi-kk/+: 24.0, TD2>kk: 23.6, PD2>kk:24.7 and psiRNAi-GD/+ 24.2, TD2>GD: 23.8, PD2>GD: 24.2). So no effect with the Pdf driver on period at all, and a small 0.4 hr effect with TD2. Moreover, no effects with 2 other RNAi lines shown in this Data set with either tim or Pdf drivers. I am not sure if the authors have more control data and if the Data set table represents only the actual screening data, but clearly additional controls are required. This is an important point, because no other behavioral phenotypes after psi knockdown could be observed (LD behavior at different temperatures and light-PRC, Figure 3—figure supplements 2 and 3).

Indeed, at first sight, it might appear that the PSI RNAi phenotypes are quite modest. Because the tim-GAL4 and pdf-GAL4 drivers cause a ca. 1hr period lengthening on their own (this has been observed since these drivers were first reported in the late 90s by the Hall lab), the only meaningful period comparison is between driver/+ flies and drivers/RNAi flies, which is what we showed on the figures. Importantly PSI came out of a screen of over 600 RNAi lines and was one of the rare genes to show a short period phenotype when downregulated, so it is clear that a 1.5 hour period shortening compared to driver control is highly significant, and specific. What the RNAi/+ control in dataset1 show is that the RNAi transgenes, on their own, do not shorten period. This is now explained more thoroughly in the manuscript. We would like to mention that in the late stages of preparing our original manuscript, a portion of the text that contained much of these explanations was inadvertently deleted. We apologize for this oversight, which probably contributed to the concerns of the reviewer.

We were however able to further support our claim that PSI regulates period. When we monitored circadian behavior at 30°C instead of 25°C, we observed that the circadian period of the timGAL4/+ controls was not significantly different from the RNAi/+ controls. We could therefore meaningfully compare both RNAi/+ and timGAL4/+ controls to the experimental flies. The results are clear, period is shorter in the experimental flies than in the controls. This was added to Figure 2C.

We also considerably increased the number of PSI overexpressing flies tested, and confirmed that period is indeed long in these flies.

Thus, we have clearly established that PSI levels are critically important for the period length of circadian behavior.

2) The authors see stronger effects with tim-gal4 (both knock-down and overexpression of psi) compared to Pdf-gal4. They aim to distinguish between potentially stronger tim-gal4 expression in the s-LNv, or a period-determining function of non-s-LNv neurons by applying a tim-gal4/Pdf-gal80 combination, which should eliminate gal4 from the s-LNv but not all the other clock cells. Unfortunately, this experiment did not allow for a clear distinction, as the period shortening after knockdown was in between that of tim and Pdf drivers alone. Overexpression with the tim-gal4/Pdf-gal80 combination recapitulated the high percentage of arhythmicity seen with tim alone, but not the period-lengthening, whereas overexpression with Pdf-gal4 only had no effect. Overall the results suggest a role for non-s-LNv neurons in setting period length and controlling rhythmicity, but I think this should be repeated with other, non-s-LNv drivers to get a clearer picture (e.g., it cannot be ruled out that Pdf-gal80 is completely blocking gal4 in the s-LNv). I suggest using the splitE cell-gal4 and drivers specific for the DN1 to solve this issue.

As described above in response to one of Reviewer #2’s comments, we tried to map arrhythmicity to specific neurons, but the results are not straightforward, and an arrhythmic phenotype is more difficult to interpret than a change in period We would thus prefer to leave these data out of the manuscript to keep the focus on the most important and solid results, but if the editors and reviewers consider it necessary for us to add these data, we will do so.

3) psi overexpression with tim-gal4 and tim-gal4/Pdf-gal80 produces very high levels of arrhythmicity and the few rhythmic flies (n=5 and n=4, respectively) have low average power values. Considering the low number of rhythmic flies and the low power values, I find it problematical to claim that overexpression with tim-gal4 causes period-lengthening. Could the authors show actograms that clearly show the longer period in these flies compared to the tim-gal4/Pdf-gal80 flies. Lacking additional support (such as convincing actograms and/or higher n's), I think it is not OK to conclude that the overexpression results are in line with the knockdown results (which are problematical in itself, see major point 1 above).

We agree that the number of rhythmic flies was low. We have repeated these experiments and considerably increased the Ns. We confirmed our initial results.

4) The peripheral clock data in Figure 3 do not look very convincing, due to the poor rhythmicity of the luciferase oscillations in DD. As correctly mentioned in the Materials and methods part, it is expected and was reported previously that rhythms of these reporters dampen rapidly in DD. But I am not sure if it is valid to calculate period values from 48 hr only, particularly if one the 2nd day the oscillations are very low amplitude. Perhaps it would be better to look in LD (where the reporters cycle with high-amplitude) to see if the rhythms in psi knock down flies are slightly phase-advanced? Or use dissected peripheral tissues in hope of stronger rhythms in DD (whole body rhythms could be further dampened due to internal desynchronization between tissues).

We have not been very successful with dissected organs in our lab in DD. In LD, the effect would be expected to be minimal based on behavior (see Figure 3—figure supplement 2), and we think it would be unlikely to observe a significant effect. However, we are confident that the results presented are solid. Phase changes are clearly visible on the graphs, and period was reproducibly shorter in experimental flies in all experiments, even though for one reporter we were slightly above a P value of 0.05. In addition, the period shortening observed with luciferase is very similar to that observed with locomotor behavior.

5) Figure 5 and accompanying Results text. I think this is the key figure about the molecular effects of reducing psi function (the effects on cwo are relatively weak and much less convincing). The data in Figure 5 clearly show that tim introns that are usually retained at cold temperatures (due to no splicing at the usual splice sites, I assume) and lead to higher mRNA levels of these particular transcripts, are also elevated at 25°C when psi is being knocked down. So it seems that psi is responsible for splicing out these introns at warmer temperatures. In contrast, an intron normally retained at high temperatures (29°C) and resulting in high tim-M transcript levels is spliced out in psi knockdown heads at 25°C, leading to lower tim-M levels both at 25°C and 29°C. This suggests that for this transcript, psi seems to promote intron retention (so to reduce splicing) at warmer temperatures. How can this be explained by a common molecular mechanism? Also, it doesn't help that the authors refer to an accompanying paper (not available for this reviewer) for the nature of the different tim transcripts and splicing events. Without a map explaining these mainly totally novel alternative tim transcripts it is impossible to follow what is going on. So, a map seems mandatory, also because the reader should not be forced to swap to a different paper in order to understand the current one.

We regret that the reviewer could not access the accompanying paper. However, we agree that a map of tim splicing should be added to our manuscript and we have done so. In terms of mechanisms for differential effects on different splicing events, it is possible that one splicing event regulates the probability of the other. We are now mentioning this possibility in the Discussion.

6) Figure 6 supports the idea that psi regulates period by tim splicing. I am bit confused by the genotype description used in the figure and Table 3. In the other parts of the paper tim-gal4 UAS-dicer2 was abbreviated as TD2, but here 'TG4/+;UAS-Dcr2/+ was used. Are these the same flies or constructs on different chromosomes. Please explain. Also, as in Figure 2, the controls for RNAi/+ are missing.

Yes, we used a UAS-Dcr2 transgene on a different chromosome, because we needed to be in a tim⁰background for the experiments shown on Figure 7. tim is on the second chromosome, so to build the flies it was easier to use a 3^rd chromosome UAS-Dcr2 insertion. This is more clearly explained in the figure legend.

[Editors' note: the author responses to the re-review follow.]

Essential revisions:

1) Figure 2C to H. At 30°C there are clear period-shortening effects (panel 2C). But from Table 3 and it's difficult for the reader to extract from these numbers whether the UAS-RNAi, and UAS-Psi constructs are significantly different from the experimentals at 25°C. Where are the UAS-Psi RNAi and UAS-Psi overexpression controls for 25°C work? Without these it's difficult to assess how valid are the period changes at 25°C. These should be included. Some of the statements being presented from Figure 2 are possibly incorrect e.g. panel 2D. The authors need to present the 25°C UAS/+ results graphically in Figure 2 and perform the appropriate statistical comparisons. In fact the UAS psi/+ result would actually strengthen their case for period lengthening for panels F and H.

We have added the PSI RNAi/+ controls to Figure 2A. They show that the RNAi lines, on their own, do not shorten period, compared to wild-type flies. In addition, the experimental flies are statistically shorter than these controls. However, the difference is smaller than against tim-GAL4, Uas-dicer2 (TD2)/+ controls, because tim-GAL4 causes a well-known ca. 0.8 hr period lengthening at 25°C. Because this period lengthening is dominant, the key comparison to understand the impact of PSI downregulation on circadian behavior is TD2/+ vs. TD2/Psi RNAi. This is the case for the PD2 combination as well, because pdf-GAL4 similarly lengthens period in a dominant manner. The effect on period is weaker when RNAi expression is restricted to PDF cells with PD2, or to non-PDF cells with TD2/-pdf-GAL80. As a result, while the experimental flies are statistically significantly shorter than PD2/+ or TD2/+; pdfGAL80/+ control flies, they are not shorter than PSI-RNAI/+ controls. This does not invalidate our conclusions that both PDF and non-PDF cells are implicated in the PSI phenotype. The presence of the RNAi transgenes, on their own, has no effect on period (new Figure 2A), and the comparison that has to be done, to evaluate the contribution of PSI-RNAi expression, is experimental flies vs. the driver controls (PD2/+ or TD2/+; pdfGAL80/+) because of their dominant effect on period. To make sure that the figure is not confusing to the reader, we only showed the key genotype comparisons in Figure 2B and C, but in the figure legend and the main text we made every effort to make clear to the reader why these are the key comparisons, and that the RNAi on their own do not impact period.

We have now also included the UAS-PSI/+ controls in Figure 2F-H. As expected, experimental flies are statistically longer than these control flies, but again, the key comparison is actually experimental flies vs. GAL4 driver controls.

2) Figure 3A and B isn't so convincing visually because there is only one cycle, even though the cosinor method used is appropriate. Might this be relegated to a supplementary figure as it adds very little to the manuscript?

As suggested, we have now moved this figure to the supplementary figures (Figure 2—figure supplement 2A, B). Please not however, that there are two cycles on the figures, not just one.

3) Further in Figure 3: It is still very difficult to see the period shortening in the bioluminescence traces (B, D). In particular it is difficult to distinguish between the black circles and black squares (RNAi/+ and test flies, respectively). It would help to at least use colored symbols to help distinguishing the different genotypes.

Thank you for this suggestion, we have improved the presentation of this figure.

4) Figure 4 does not add much to this manuscript, where two cwo splice forms appear to be reduced and one is increased. The results are described briefly then immediately dropped – this could be relegated to a supplementary figure.

As suggested, we have moved this figure to the supplementary figures (Figure 3—figure supplement 1).

5) The tim splicing results are very confusing and need to be clarified. The cold tim isoforms (tim-cold and tim sc) are elevated at warm temperatures in the psi mutant so normally psi would enhance splicing at cold temperatures. The warmer tim isoform (tim-M) is reduced at 25°C in the psi mutant so we'd presume that psi normally enhances this isoform at warm temperatures. Therefore psi normally has opposite effects on the tim isoforms, so a general temperature-sensitive psi mechanism appears to be excluded.

We have made additional efforts in the text (Results and Discussion) to make clear the impact of PSI on tim splicing. Briefly, PSI promotes splicing favored at warm temperature, while inhibiting those favored at cold temperature, and it does this at any temperature. As we discussed, while PSI determines the ratio of tim isoforms, their temperature sensitivity might be encoded through splice site strength, as for per 3’UTR intron retention event. In the Discussion, we proposed that splicing events might be co-regulated, and that therefore favoring one can, at the same time, indirectly influence the probability of another (excerpt from the previous Discussion: “Another interesting question is how PSI affects differentially specific splice tim isoforms. One possibility is that the execution of a specific tim splicing event influences that of another”). We have expended this point of Discussion to make it clearer. We believe that with this addition and a few minor text corrections in the relevant Results section, the impact of PSI on tim splicing should be entirely clear to the reader.

6) Figure 4—figure supplement 1 the UAS RNAi controls are not shown here. Are they temperature compensated? The Td2 control is not temperature compensated and has a relatively large change in period at hot temperature.

They are temperature compensated. We had left them out because of the dominant impact of TD2, but we have now added them.

7) 'Collectively, these results indicate that PSI shifts the balance toward a warm temperature tim RNA isoform profile at an intermediate temperature (25°C).' This sentence makes no sense – no general mechanism can emerge as PSI appears to have opposite effects on the cold/warm tim isoforms. It enhances the warm forms and reduces the cold forms. Do the authors mean wild-type PSI or the psi-mutant? Please clarify.

Please see our response above on the possibility that a splicing event influences another. Also, we have added “in wild-type flies”. We hope that this sentence now makes sense.

Author details

1. Lauren E Foley

   Department of Neurobiology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7635-7338

2. Jinli Ling

   Department of Neurobiology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Conceptualization, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Radhika Joshi

   Department of Neurobiology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Naveh Evantal

   Hebrew University of Jerusalem, Jerusalem, Israel

   Contribution

   Resources, Writing—review and editing

   Competing interests

   No competing interests declared

5. Sebastian Kadener

   1. Hebrew University of Jerusalem, Jerusalem, Israel
   2. Brandeis University, Waltham, United States

   Contribution

   Resources, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0080-5987

6. Patrick Emery

   Department of Neurobiology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   Patrick.Emery@umassmed.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5176-6565

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