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

Reviewer #1:

[…] My only comments refer to the text.

1) Abstract "However, mechanisms underlying such delays are unclear." I would say that the mechanisms are not "unclear" but that "our understanding of the mechanisms involved is incomplete" since there is clear evidence (mentioned in the manuscript, for instance, in the Introduction final paragraph) that phosphorylation, protein stability, and dimerization of PER and TIM, all contribute to the functioning and period-setting of the clock.

2) Results section: "Overall, these findings strongly suggest that the free-running circadian component of the prp4-knockdown phenotype maps to LNvs." Rather than "maps" I would say, "the function of prp4 in LNvs contributes to setting the periodicity of the clock". This because the knocking down prp4 in all clock neurons (Figures 1A, B) is not quite the same as knocking it down only in LNvs (Figures 1D-G).

3) Discussion section: "Importantly, our findings address a longstanding question in the circadian field, specifically how negative feedback by clock proteins is delayed in order to permit distinct phases, and therefore oscillations, of transcriptional activation and repression." Rather than "address" I would say, "contribute to our understanding of".

For points 1-3, the text of the manuscript was revised as suggested.

4) In the Introduction and Discussion I think it is appropriate to cite the work of Beckwith et al., 2017 (10.1534/genetics.117.300139), which shows that alternative-splicing regulator, SR-related matrix protein of 160 kDa (SRm160), affects gene expression in pacemaker neurons.

The following sentence was included in the Discussion: “In Drosophila, the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) modulates PER levels locally in the pacemaker neurons to regulate circadian rhythmicity (Beckwith et al., 2017)”.

5) In the Discussion I think it is worth mentioning another situation where a specific alternative splicing event has an impact on clock function, namely the BK potassium channel, where inclusion of a specific exon changes the electrophysiological properties of the channel, most likely contributing to the circadian changes in firing rate of SCN (work from Andrea Meredith's lab).

The following sentence was included in the Discussion: “Alternatively, splicing mechanisms could regulate diurnal rhythmicity of neuronal excitability, as proposed for splicing of BK channels in the suprachiasmatic nucleus (Shelley et al., 2013).”

Minor Comments:

Difficult to resist suggesting that rather than "tim-tiny", this intron be called "Tiny tim"….

Additional data files and statistical comments:

The most relevant data (RNA-Seq data) have been made available (Gene Expression Omnibus standard repository, accession # GSE115163).

Reviewer #2:

[…] The data look really convincing although I will have a couple of suggestions.

– Results section and Figure 1: complex behavioral rhythms are obtained when prp4 is downregulated in PDF cells whereas long period rhythms are obtained with all-clock neurons downregulation (Figure 1). To me, it suggests that prp4 downregulation has also effects in pdf- oscillators, which is no surprise. I would not simply conclude that the prp4-knockdown phenotype maps to LNvs.

We agree that the circadian effects of prp4 are unlikely to be mediated exclusively by sLNvs. However, because the pdf-selective downregulation of prp4 can drive altered circadian behavior, we conclude that pdf+ clock cells contribute to setting the periodicity of the clock. As per Reviewer 1’s suggestion, we modified the text to “Overall, these findings suggest that the function of PRP4 in LNvs contributes to setting the periodicity of the clock” instead of "Overall, these findings strongly suggest that the free-running circadian component of the prp4-knockdown phenotype maps to LNvs.”

Additionally, we modified the sentence to: “We identified PRP4 in a screen for novel regulators of the free-running circadian period, and established that PRP4 is necessary in clock tim+ cells, to maintain 24-hour period and robust rhythmicity of the circadian clock.”

– Subsection “PRP4 is required for robust TIM and PER cycling” and Figure 2: the PER phosphorylation phenotype is strong (Figure 2D). The authors propose that it is a consequence of a defect in PER nuclear transfer, due to TIM decrease. I think that the possibility of a direct effect of prp4 on PER should be tested by comparing PER phosphorylation in prp4+ and prp4 RNAi flies with a tim⁰ genetic background. I acknowledge that it might not be easy to see since TIM affects PER phosphorylation, but I believe that it is an important point to clarify.

Would it be possible to analyze the effects of a similar decrease of TIM (50% decrease at ZT18-20 based on Figure 2E) but as a consequence of another molecular defect, on PER phosphorylation?

This is a very good point. To better acknowledge that prp4-dependent PER phosphorylation phenotype could be independent of prp4 effects on TIM, we added the following sentence to the Results section: “Alternatively, prp4 depletion could have a TIM-independent effect on PER to regulate its phosphorylation profile.”

It would be very difficult to examine effects of prp4 on PER phosphorylation in a tim⁰ background because loss of TIM alone dramatically reduces levels of PER and affects PER phosphorylation, which would likely obscure effects of prp4 (Price et al., 1995). We considered the possibility of examining a 50% decrease in TIM by using tim⁰/+ flies, but as these flies do not have a behavioral phenotype, they would not be relevant in terms of explaining the prp4 phenotype. We believe that reduction of TIM in flies with prp4 downregulation is greater in sLNvs than across the rest of the brain. Nevertheless, it is true that some aspects of the prp4 phenotype might be independent of TIM, so it is best to acknowledge this as we propose to do above.

– Subsection “PRP4 regulates tim splicing”: It is unclear to me how the 45 genes exhibiting differential splicing (Supplementary file 4, which I could not find) compare to the 5 genes that are described in Figure 3A. Is it the different analysis method? Please clarify.

To clarify the methods used to analyze the RNA-Seq data, we changed the sentence to “Using the Comprehensive AS Hunting (CASH) method, which assays for splicing events, our analysis identified 45 genes exhibiting differential splicing upon prp4 downregulation with FDR ≤ 0.05 (Wu et al., 2017) (Supplementary File 3).” To further narrow down relevant targets, we utilized additional algorithms and identified overlapping splicing events. We state this point as “Our initial splicing analysis was performed with CASH, but we additionally ran the Cufflinks-2.2 pipeline to obtain psi (percent spliced in) information for differentially spliced isoforms (Supplementary File 4).” The 5 genes described in Figure 4A are the overlapping targets identified by both of the splicing algorithms. We believe the legend of Figure 4 clearly makes this point: “(A) Only 5 genes were identified as differentially spliced upon prp4 downregulation with both CASH (Wu et al., 2017) and Cufflinks/differential psi (percent spliced in) pipelines (Trapnell et al., 2010; Trapnell et al., 2013).”

– Subsection “PRP4 regulates tim splicing” third paragraph and Figure 3 (+ Figure 4—figure supplement 2): I understand that tim-tiny mRNA is increased by two-fold in prp4 RNAi flies (Figure 3D). However, I suppose that the relative quantity of tim-tiny mRNA in comparison to total tim mRNA is a key parameter when it comes to explaining the effects of splicing regulation on total TIM protein levels. Please clearly indicate in the text what fraction of total tim mRNA tim-tiny represents in wild type flies.

It seems to me the effects of the different tim cDNAs on TIM protein levels (S2 cells and transgenic flies in Figure 4) are difficult to extrapolate to wild type and prp4 RNAi flies if this ratio is not known. Does Figure 4—figure supplement 2B indicate that the spliced and retained forms are present in similar amounts?

Figure 5 and 6: I understand that the tim-tiny/tim ratio is indeed close to 1 in average. If I understand this well, that would say that half of tim transcripts do not produce a functional TIM protein. I believe that this point should be emphasized in the paper even before telling about the cycling of this ratio.

We agree that the ratio of isoforms that contain tim-tiny (tim-RM and tim-RS) to those that do not contain this intron is an important parameter to highlight in our study. We revisited our Cufflinks-2.2 analysis of the RNA-Seq data to better calculate this ratio (provided in the new Supplementary File 5). This analysis indicated that the isoforms that retain tim-tiny are twofold more abundant than isoforms that do not contain it (at ZT 8 when all of the RNA-Seq samples were collected). This finding would imply that at ZT8 the majority of tim isoforms do not produce full-sized TIM or produce unproductive protein that is rapidly degraded (Figure 5). As our data indicate, the ratio of spliced and unspliced forms changes over time (Figure 6A-B). The new information about the ratio is included in the following sentence: “To estimate how common tim-tiny retention was in control flies (GMR/+), we quantified the ratio of isoforms that contain tim-tiny (tim-RM and tim-RS) to those that do not contain this intron using our Cufflinks-2.2 output (Supplementary File 4). This analysis indicated that the isoforms retaining tim-tiny were twice as abundant as other isoforms (at ZT 8 when all of the RNA-Seq samples were collected) (Supplementary File 5).”

Minor Comments:

– The title does not tell much about the mechanism and I would suggest to make it more specific. For example, something in this line: Spliceosome Factors Target timeless mRNA to control protein accumulation in the Drosophila circadian oscillator.

To incorporate the effect on circadian behavior, we have changed the title to: “Spliceosome Factors Target timeless mRNA to control protein accumulation and circadian behavior in Drosophila”

– Zheng and Sehgal, 2012 (Introduction section) does not seem to be in the list of references

The reference was added.

– Figure 2 legend: E is quantification from D rather than A?

The figure legend was corrected.

Reviewer #3:

[…] The major unresolved conceptual issue is how the differential splicing of this intron is produced throughout the day by the clock. The authors speculate that daily changes in the level of splicing factors (and there is some evidence for this in earlier tissue-specific RNA profiling analyses) might produce the altered intron inclusion. Since prp4 is a kinase, there might also be circadian regulation of its kinase activity. This possibility could be mentioned in the Discussion.

The following sentence was added to the Discussion: “In addition to changes in total levels of PRP4, circadian regulation of its kinase activity could contribute to differential splicing of tim over the course of the day.”

Minor Comments:

1) Two lines on Table 1 are both labeled TUG>Dcr2;prp4 RNAi (GD), but one of these (probably the second one) should perhaps be labeled KK instead of GD. Do these lines target different regions of the prp4 mRNA to show the effects are target specific? And why does the "stronger" KK line have less effect on period length and rhythmicity than the GD line (Figure 1F and G)?

The second line was corrected to “TUG>Dcr2;prp4 RNAi (KK)” in Table 1.

With respect to the strength of the KK line, we believe the reviewer is referring to the non-significant effect of KK on period with the pdf driver. However, please note that a very small percentage of these flies have a single rhythm that could be analyzed for period, as opposed to flies with GD driven by pdf (Figure 1E-F). We consider the KK line ‘stronger’ than GD because KK has a longer average period and less rhythmicity than GD when driven with the TUG driver (Figure 1B, Table 1).

The GD line (VDRC:27808) and KK line (VDRC:107042) target distinct, but overlapping regions of prp4.

2) Define the differences between the boxes and error bars on Figure 1.

The boxes extend from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. Whiskers extend from the lowest to highest value. Since the same description would be relevant for Figure 5D, we updated the legends of both Figure 1 and Figure 5.

3) While the text states that the morning peak of activity is triggered by lights in Figure 1C, there still seems to be some anticipation – just less than in the control.

The text was adjusted to “Compared to controls, the flies with circadian-cell-specific prp4 knockdown (TUG; Dcr2>prp4^RNAi (GD)) exhibited a delayed evening activity peak as well as a slightly delayed morning peak”

4) The complex rhythm in Figure 1D seems to include an ~6hr phase shift at the end of the record. Please comment on this in the text. The figure legend indicates that these rhythms are double-plotted but they appear to be single-plotted.

The reviewer is correct in that there appears to be a phase shift (delay or advance) in many records. We have modified the sentence to read: “These complex rhythms were generally characterized by changes in the behavioral pattern, often manifest as phase shifts during day 4 or 5 of constant darkness (see ~6-h shift in the record shown), in the midst of an otherwise rhythmic record (Figure 1D).”

We apologize for the inaccuracy and have deleted the word ‘double-plotted’.

5) Why are the intervals for the collection of samples for TIM analysis in Figure 2E 2hr long while they are precise times for PER analysis?

There was no strategic reason for this. For the TIM analysis, we pooled data from multiple experiments in which samples were collected at similar, but not identical, times so we indicated the collection times as intervals.

6) There does not seem to be a Supplementary file 3 nor a Supplementary file 4.

These files will be uploaded with the final paper submission.

7) The model presented in Figure 6 argues that the delayed accumulation of TIM during the day due to inclusion of the extra intron (which truncates the reading frame of the protein) introduces a phase delay into the clock. But overexpression of cDNA with this intron missing produces longer periods, which apriori is inconsistent with the model (it should speed the clock, not slow it). Presumably the overexpression is producing some additional effects like prolonged repression of the E box, outside the confines of the model. Some discussion of this point is needed.

Overexpression of tim cDNA from the UAS promoter typically results in longer periods as it does not allow for feedback and so leads to massive accumulation of TIM (Yang and Sehgal, 2001). Please note that earlier studies showing period-shortening with PER over-expression used constructs that included a per promoter that could be repressed by feedback (discussed in Yang and Sehgal, 2001). It is true that we do not see a period difference with ‘tim-retained’ and ‘tim-spliced’ overexpression constructs (Figure 5D), which may be because these constructs have small effects in a wild type background. However, the reviewer is absolutely correct in that the “intron missing” form should produce shorter periods than the ‘tim-retained’ form, and we now show the data for expression of these constructs in a tim⁰ background (included as Figure 5E-F; also shown below). Note that both forms rescue with a longer period, as expected of UAS constructs, but rescue with the spliced form (‘tim-spliced’) yields a shorter period, albeit in a smaller percentage of flies. The latter point of lower rhythmicity also supports our idea that delays are required for a robust oscillator.

We added the following sentences to the legend of Figure 5: “(E) TUG-driven expression of tim cDNA, with both ‘tim-spliced’ and ‘tim-retained’ constructs, rescues tim⁰ circadian rhythmicity. n.s., not significant at the 0.05 level, **p ≤ 0.01, ****p ≤ 0.0001 by pairwise Fischer’s exact test (n=28-41). (F) TUG-driven rescue of tim⁰ circadian rhythms with tim cDNA that includes tim-tiny (tim⁰,TUG>tim-retained) lengthened the period compared to the rescue with tim cDNA lacking tim-tiny (tim⁰,TUG>tim-spliced). ***p ≤ 0.001 by Student’s t test, n=10-22.”

These findings were summarized in the Results section as “To further understand the effect of tim-tiny splicing on circadian behavior, we overexpressed ‘tim-spliced’, ‘tim-retained’ and ‘tim-retained-ssM’ using the TUG driver in the tim⁰ homozygous background. We hypothesized that splicing of tim-tiny is necessary for the maintenance of circadian rhythmicity and so the ‘tim-retained’ construct would rescue the behavioral rhythmicity most efficiently because it allows for both the splicing and retention of the intron. Additionally, because prp4 knockdown increases the retention of tim-tiny (Figure 4) and prolongs the rhythm (Figure 1), we speculated that the ‘tim-spliced’ construct would rescue with a shorter period than the ‘tim-retained’ construct. As expected, the expression of tim cDNA with a 5’ splice site mutation in tim-tiny (‘tim-retained+ssM’) did not restore rhythms in tim⁰ flies (Figure 5E). The other two cDNA constructs, ‘tim-retained’ and ‘tim-spliced’ rescued the rhythmicity in 54% and 32% of tim⁰ flies, respectively. Importantly, there was a significant difference in period length between the flies that were rhythmic (Figure 5F). As discussed above, the UAS-GAL4 system typically over-expresses proteins and so rescues per/tim mutants with longer periods (Yang and Sehgal, 2001), which we observed for the ‘tim-retained’ isoform (~26 hours). Shorter periods were seen with the ‘tim-spliced’ version (Figure 5F), supporting the idea that tim-tiny retention promotes clock delays.”

We changed the title of Figure 5 to “Intron retention in tim decreases TIM levels and affects circadian behavior” to better convey the message of the new panels.

Additional data files and statistical comments:

Supplementary files 3 and 4 are missing and are important. They need to be provided. The statistical analysis is fine.

The files will be uploaded.