To determine whether circRNAs can influence neuronal differentiation, we initially mapped the circRNA inventories at different stages of neuronal differentiation and compared these to other available circRNA datasets of neuronal origin from mice and humans (Rybak-Wolf et al., 2015). Identification of circRNAs from RNA-seq experiments has often been based on quantification of relatively few reads across the circRNA backsplicing junction (circBase Glažar et al., 2014), and current circRNA prediction algorithms inevitably lead to the calling of false positives (Hansen et al., 2016; Jeck and Sharpless, 2014). Hence, to immediately validate the circular nature of to-be called circRNAs, we first performed standard rRNA depletion and subsequently either included or excluded RNase R treatment step prior to RNA-sequencing. Specifically, we used an established differentiation model for CNS-type glutamatergic neurons, based on E14 mouse embryonic stem cells (mESCs) that reportedly yields a purity of glutamatergic neurons of >90% (Bibel et al., 2007). RNA was isolated from three stages of differentiation, mESCs, neuronal progenitor cells (mNPCs) or neuronal differentiation day 8 (mN8) and rRNA depleted (+/- RNase R) prior to library preparation and RNA-seq (Figure 1A). Successful differentiation at the mNPC and mN8 stages was confirmed by the appearance of elongated intercellular dendritic extensions (mN8) (Figure 1—figure supplement 1A) and robust upregulation of several classical neuronal markers including, Ntrk2, Map2, and Tubb3 (mNPC and mN8), while stem cell pluripotency marker Nanog became significantly reduced upon differentiation (Figure 1—figure supplement 1B). Using available circRNA prediction tools CIRI2 (Gao et al., 2018), find_circ (Memczak et al., 2013) and CIRCexplorer2 (Zhang et al., 2016) on the non-RNase R-treated RNA, we identified between 792–1167 circRNAs in mESC, 2230–2893 circRNAs in NPC and 1902–2316 circRNAs in differentiated neurons at mN8 stage (Figure 1B). Upon RNase R treatment most circRNAs either remained unchanged or became enriched, but a considerable fraction of the predicted circRNAs in mESC, mNPC, and mN8 preparations, became depleted by the 3’−5’ exonuclease (CIRCexplorer2: 19.5–36.5%; CIRI2: 7.2–16.6%; find_circ: 38.7–52.3% depleted) (Figure 1—figure supplement 1C). All prediction algorithms showed a correlation between expression level and RNase R resistance, suggesting that mostly low-count circRNAs candidates are likely false positives (Figure 1—figure supplement 1D). From a total of 3581 enriched circRNAs after RNase R treatment (all stages), 1449 circRNAs overlapped between all three circRNA prediction algorithms, and this subset represents a high-confidence circRNA inventory (Hansen et al., 2016; Figure 1C and Supplementary file 1). We next assessed the circular-to-linear ratio of identified circRNAs (find_circ), by comparing splice site usage in circular vs. linear splicing events (Memczak et al., 2013; Rybak-Wolf et al., 2015). This analysis revealed vast differences in the steady-state levels of these isoforms and demonstrated that many circRNA species are considerably more abundant than their linear precursors (Figure 1D). Confirming previous results (Rybak-Wolf et al., 2015), introns flanking the circRNAs are generally longer than average introns and circRNAs often tend to cluster at the 5’ end of their respective precursor RNA (Figure 1—figure supplement 1E–F). Our results suggest that in order to obtain high confidence circRNA inventories from RNA-seq data, it is beneficial to use multiple circRNA prediction algorithms and to enrich for bona fide circRNAs, by depletion of linear RNAs using RNase R.

Figure 1 with 1 supplement see all

Download asset Open asset

We next tested differential circRNA expression during differentiation, which revealed marked changes in circRNA expression over the 16-day timecourse (Figure 1E; left panel). Kmeans clustering of circRNAs by expression (Top 100 highest expressed) pattern showed two main clusters with peak expression at mNPC and mN8 (Figure 1E; right panel). Comparison with previously identified mouse and human homologue circRNAs, isolated from mouse brain regions or cell lines of either murine or human origin (Rybak-Wolf et al., 2015), revealed a substantial overlap between circRNAs at differentiated stages (e.g. 80% of all 1449 circRNAs found in differentiated murine p19 cells and primary neurons, 45% of Top100 found in human SH-SY5Y and 75% overlap with circRNAs found in the human ENCODE data previously analyzed Rybak-Wolf et al., 2015; Stagsted et al., 2019; Figure 1—figure supplement 1G). We confirmed differential expression of a subset of the most abundant and upregulated circRNAs (circTulp4, circMagi1, circRmst, circEzh2, circHdgfrp3, circZfp827, circMed13l, circZfp609, circSlc8a1, circNfix) using RT-qPCR with amplicons across the backsplicing junction (Figure 1F–G). 75% of the top-100 expressed mouse circRNAs was also found in human circRNA datasets (Rybak-Wolf et al., 2015; Figure 1—figure supplement 1G). We conclude that significant changes in circRNA expression patterns are induced upon neuronal differentiation and that the majority of these circRNAs are conserved between various neuronal cell-types originating from humans and the mouse.

To ascertain whether the highly upregulated circRNAs might contribute to the process of neuronal differentiation, we next depleted a number of candidate circRNAs by RNA interference. We first tested knockdown efficiency of circZfp827 (circZNF827 in humans) by lentivirally delivered dishRNAs (Kaadt et al., 2019) targeting the backsplicing junction in either mESC, p19, SH-SY5Y or L-AN-5 cells, of which the latter three cell lines are well-established models of neuronal differentiation following retinoic acid treatment. Knockdown efficiency in mESC and p19 proved relatively poor (30–60% remaining circRNA) compared to the two human cell lines: SH-SY5Y (10% remaining) and L-AN-5, which displayed superior results (<8% remaining) (Figure 2A and Figure 2—figure supplement 1A). Moreover, when testing SH-SY5Y cells for an increase of neuronal differentiation markers NTRK2, NEFL, MAP2 and TUBB3 upon retinoic acid treatment, only NTRK2 was significantly upregulated upon differentiation (Figure 2—figure supplement 1B), whereas these genes showed a more expected and dynamic expression pattern in L-AN-5 cells (Figure 2B). We therefore transduced L-AN-5 cells with lentiviral dishRNA vectors to perform knockdown of 14 candidate circRNAs (Figure 2A and Figure 2—figure supplement 1C; circTULP4, circSLC8A1, circZNF609, circHDGFRP3, circMAGI, circRMST, circZNF827, circANKIB1, circMED13L, circCDYL, circUNC79, circHIPK3, circNFIX, circCAMSAP1) (Supplementary file 2a) and subsequently subjected these to retinoic acid-induced differentiation followed by neuronal marker quantification in order to probe for changes in differentiation. In general, we observed efficient knockdown (Figure 2A and Figure 2—figure supplement 1C). While the majority of knockdowns did not significantly change neuronal marker expression, knockdown of circZNF827 (and to a lesser extent circANKIB1), produced a significant and reproducible increase in neuronal marker expression upon differentiation (Figure 2B and Figure 2—figure supplement 2A). Importantly, the linear ZNF827 mRNA was not affected by backsplicing junction-specific knockdown (Figure 2—figure supplement 2B). The upregulation of neuronal markers following circZNF827 knockdown was also evident at the protein level for MAP2 and TUBB3 (Figure 2C, and quantified to the right). In addition, proliferation assays demonstrated a smaller S-phase population (32% to 24%) upon circZNF827 knockdown, suggesting lowered replication kinetics (Figure 2D and Figure 2—figure supplement 3A–B). This phenomenon was accompanied by a minor stall in G₂/M phase, while G₀/G₁ phase was not significantly affected between control and circZNF827 knockdown. Taken together, our results suggest that circZNF827 knockdown exerts a repressive effect on proliferation, while it enhances neuronal marker expression and hence differentiation.

Figure 2 with 3 supplements see all

Download asset Open asset

We next asked whether the retinoic acid receptors (RARs), which represent central nodes in relaying anti-proliferative differentiation cues during neuronal development (Gudas and Wagner, 2011), and are key targets of retinoic acid (RA), also become upregulated upon knockdown of circZNF827. Indeed, knockdown of circZNF827 leads to a moderate but significant increased expression (1.5–2.5 fold) of RARB and RARG, while RARB remained constant (Figure 3A). Since most circRNAs have been reported to predominantly localize in the cell cytoplasm, we addressed the localization of circZNF827, circANKIB1 and circTULP4 by cellular fractionation. These circRNAs are mainly cytoplasmically localized in L-AN-5 cells (~90% cytoplasmic signal) (Figure 3B). We therefore hypothesized that circZNF827 could potentially affect RAR-mRNA stability post-transcriptionally in the cell cytoplasm. However, BrU pulse-chase mRNA decay assays demonstrated no significant change in RAR-mRNA decay rates upon knockdown of circZNF827 (Figure 3C). Next, we investigated transcription rates, by treating cells with a short pulse of BrU, followed by BrU immunoprecipitation to quantify de novo labeled RNA, serving as a proxy for transcription rates during control- or knockdown of circZNF827. As expected from the constant mRNA decay rates, BrU incorporation was moderately upregulated, although significantly only for RARA, upon circZNF827 knockdown (Figure 3—figure supplement 1). Although this moderate increase in transcription measured at a single late time-point, cannot readily explain the larger effect on the RARA mRNA level (2.5-fold) measured at steady-state, our results suggest that circZNF827 contributes to controlling the RARA receptor levels in order to keep neuronal differentiation in check.

Figure 3 with 1 supplement see all

Download asset Open asset

Our results indicate that L-AN-5 cells are lowering their proliferation rates and promote RAR-signaling by transcriptional upregulation of one of these transcription factors when circZNF827 levels are low. To test how circZNF827 knockdown affects other key factors of the neuronal transcriptome, we next performed Nanostring analyses using a neuro-differentiation/pathology panel of 760 genes with RNA purified from differentiated or non-differentiated L-AN-5 cells. 135 genes become differentially expressed (9 upregulated and 126 downregulated, fold change > +/- 2, p<0.05) due to circZNF827 knockdown after differentiation (Figure 4A, Supplementary file 2b). In line with a potential negative regulatory function of circZNF827 on neuronal differentiation, GO-term analyses show enrichment of terms including axon/dendrite structure, neural cytoskeleton, transmitter synthesis, neural connectivity, growth factor signaling and trophic factors among differentially expressed genes (Figure 4B). The most significantly upregulated gene is nerve growth factor receptor (NGFR), which plays a central role in regulating neuronal differentiation, death, survival, and neurite outgrowth (Yamashita et al., 1999; Zhu et al., 2012). Conversely, Phosphatase and tensin homolog (PTEN), STAT3 and NAD(P)H quinone dehydrogenase 1 (NQO1) were all significantly downregulated upon circZNF827 knockdown (2–4 fold), which reportedly also contributes positively to neuronal differentiation (Lyu et al., 2015; Ma et al., 2017), and in case of the latter, also renders cells more susceptible to energetic and proteotoxic stress (Hyun et al., 2012). Since NGFR is a key regulator of neuronal differentiation and the highest upregulated gene upon circZNF827 knockdown, we next focused on the mechanism of its upregulation. Using both RT-qPCR and western blotting, demonstrated a strong upregulation at both the protein and mRNA level (Figure 4C and Figure 4—figure supplement 1). This upregulation was not due to changes in mRNA decay rates, since BrU pulse-chase mRNA decay assays yielded nearly identical mRNA half-lives upon circZNF827 knockdown (Figure 4D). To address whether the observed changes in gene expression are elicited at the transcriptional level, we subjected cells to a short BrU-pulse prior to BrU immunoprecipitation and Nanostring hybridization. Interestingly, NGFR and also ATP8A2 proved to be highly upregulated (~4–6 fold) at the level of transcription (Figure 4E–F), while only NQO1 and not PTEN and STAT3 exhibited significantly reduced transcription activity (ranging from ~1.3- to ~4-fold) (Figure 4E). Also, the MAP2 gene did not change its de novo RNA output, suggesting that the tuning of the steady-state levels of PTEN, STAT3 and MAP2 mRNAs, as initially observed (Figures 2B and 4A), are mainly facilitated by posttranscriptional changes to mRNA stability. If circZNF827 is involved in a direct transcription-associated complex that regulates NGFR output, the transcriptional effects elicited by circZNF827 knockdown would require nuclear knockdown of the circRNA. Indeed, the use of dicer-independent shRNA (dishRNA) vectors proved very efficient in depleting nuclear circRNA (Figure 4G).

Figure 4 with 1 supplement see all

Download asset Open asset

Next, we assayed the cellular impact of NGFR upregulation upon circZNF827 knockdown. To this end, we NGF-treated L-AN-5 cells subjected to either control or circZNF827 knockdown, and quantified downstream signaling output by quantification of FOS, which is a well-known downstream ‘immediate early’ target of NGFR signaling. FOS levels increased significantly, strongly indicating that the higher levels of NGFR protein indeed leads to functional increase in NGFR signaling (Figure 4H and Figure 4—figure supplement 1), which can at least in part explain the upregulation of neuronal markers. Taken together, we conclude that circZNF827 serves to keep neuronal differentiation ‘in check’ by limiting expression of, and signaling by, RARA and NGFR.

To address the mechanism by which these transcriptional and post-transcriptional events are controlled by circZNF827, we next sought to identify its protein interactome. To this end, we synthesized biotin-labeled circZNF827 (linear version) and control RNAs (circTULP4, circZNF609, circHDGFRP3 and circSLC8A1) in vitro and subjected these to pull-down experiments using L-AN-5 cell lysates and streptavidin-coupled magnetic beads as previously described (Seitz et al., 2017). Silver-stained SDS-PAGE gels of retained proteins revealed unique profiles, suggesting that specific proteins exhibited increased affinity toward circZNF827, although prominent RNA-binding proteins common to both control RNAs and circZNF827 could also be observed (Figure 5—figure supplement 1A). By subjecting pulled-down fractions to LC-MS/MS, we identified several circZNF827-specific proteins, including hnRNP K and -L, while others (e.g. DHX9 and DDX3X) bound strongly to any of the bait RNAs (Figure 5A). To validate these interactions, we performed RNA-immunoprecipitaiton (RIP) using monoclonal anti-hnRNP K or -L antibodies followed by qRT-PCR across the backsplicing junction, and observed a significant enrichment of circZNF827 compared to IgG controls (>100 fold), suggesting that these interactions can be recapitulated in L-AN-5 cells (Figure 5B). As expected for these highly expressed RNA-binding proteins, both proteins associate with GAPDH mRNA, but in the case of hnRNP L, the IP/input ratios were ~18 fold higher for circZNF827, whereas hnRNP K displayed a similar enrichment of GAPDH mRNA as of circZNF827 (Figure 5B). Scrutinizing the circZNF827 sequence for putative-binding sites for hnRNP K and -L using eCLIP datasets (ENCODE consortium), proved unfeasible due to low expression levels of the ZNF827 gene in the K562 and HepG2 cells used by ENCODE. Using RBPmap (Paz et al., 2014), which is based on established RBP consensus binding sequences, revealed a potential high-affinity cluster for primarily hnRNP L binding and one site for hnRNP K in the most 3’ part of the circle-encoding sequence (Figure 5—figure supplement 1B–C). According to circZNF827 secondary RNA structures predicted by Mfold, these binding sites are located into mostly single-stranded regions within the circRNA, consistent with the binding preferences of most hnRNP proteins toward single-stranded RNA (Figure 5—figure supplement 1D). To further characterize these interactions, we prepared a stable HEK293 Flp-In T-rex cell-line expressing circZNF827 (Figure 5—figure supplement 1E–F) under the control of a tetracycline-inducible promoter (tet-on), based on the laccase vector system (Kramer et al., 2015). We then performed RIP by immunoprecipitation of endogenous hnRNP K or -L and observed a remarkable enrichment of exogenous circZNF827, compared to control IgG or GAPDH mRNA (Figure 5C and E). hnRNP L gave a particularly high IP/Input ratio (>200 fold enrichment over IgG), consistent with the results from: (1) the L-AN-5 RIP, (2) the pull-down LC-MS/MS experiment and (3) the prediction of several hnRNP L binding site clusters in circZNF827. To test whether the predicted hnRNP K/- L binding sites in the 3′ part of circZNF827 are indeed sites of interaction, we constructed a deletion mutant that removes the putative binding sites (Figure 5—figure supplement 1C, dark grey letters and Figure 5—figure supplement 1D, dotted dark grey line) and prepared HEK293 stable cell lines (Figure 5—figure supplement 1E–F). Although this mutant was expressed at somewhat lower levels than WT circZNF827, it remained virtually unbound by hnRNP K/- L similar to IgG pull-down efficiency (Figure 5C).

Figure 5 with 1 supplement see all

Download asset Open asset

We conclude that both hnRNP K and -L can be found in complex with endogenous or exogenous circZNF827 in both L-AN-5 and HEK293 Flp-in T-Rex cells, likely via high-affinity binding sites in the 3′ part of the circular RNA.

hnRNP K is a well-documented transcriptional regulator (Moumen et al., 2005; Thompson et al., 2015) that is reported to interact directly with hnRNP L and -U (Havugimana et al., 2012; Kim et al., 2000; Wan et al., 2015) and bind both DNA and RNA (Tomonaga and Levens, 1995). To assess interactions between hnRNP K, -L and -U, and their potential dependence on circZNF827, we performed co-immunoprecipitation experiments using FLAG-tagged hnRNP K, -L, and -U, and subsequently probed for their interaction with endogenous proteins (Figure 5D) in HEK293 Flp-in cells either overexpressing circZNF827 or not. hnRNP K co-immunoprecipitates both hnRNP U and hnRNP L (long isoform), but these interactions remain unaffected by increased expression of circZNF827 (Figure 5D). In accordance with these findings, immunoprecipitation of endogenous hnRNP K and -L proteins in HEK293 Flp-in cells, confirmed that a hnRNP L/hnRNP K complex can be detected although only a small fraction of the total hnRNP K/- L populations co-immunoprecipitate the other (Figure 5E, left). This complex was not affected by overexpression of circZNF827 (Figure 5E, right). Hence, exogenous circZNF827 likely does not regulate bulk hnRNP K/L-complex assembly/disassembly in HEK293 cells per se.

To test whether circZNF827 potentially regulates the normal subcellular distribution of hnRNP K, L-AN-5 cells were fractionated during control or circZNF827 knockdown and lysates subjected to western blotting. We observe a small but significant and reproducible increase in the cytoplasmic population of hnRNP K upon circZNF827 knockdown, suggesting that circZNF827 retains, albeit a very small fraction of the hnRNP K population, in the nucleus (Figure 5F). To address this, we overexpressed circZNF827 and monitored hnRNP K and L localization by immunofluorescence in HEK293 cells. Induction of circZNF827 led to accumulation of hnRNP K and to a lesser extent hnRNP L in multiple distinct nuclear foci that were not detected in control cells (Figure 5G, white arrows). Taken together, our results suggest that while bulk hnRNP K and L complex formation is not affected by circZNF827 levels, overexpression of the circRNA induces specific nuclear localization of hnRNP K and L.

Could a circZNF827-dependent hnRNP K/L-containing nuclear complex regulate the output from the NGFR gene? If such a complex is instrumental in repressing NGFR, we predict that knockdown of any of these factors would enhance NGFR expression. In support of a role in hnRNP K-mediated regulation of NGFR, it was recently reported that hnRNP K knockdown strongly induces NGFR expression in mouse ES cells (Thompson et al., 2015). To test this in our context, we designed dishRNAs for hnRNP K and -L, transduced L-AN-5 cells and assayed for NGFR expression by qRT-PCR or Western blotting. Both knockdowns increased NGFR expression at both the mRNA and protein levels, similar to the effect of depleting circZNF827 alone (Figure 6A–B and Figure 6—figure supplement 1A–B). However, co-depletion of circZNF827 with any of these factors strongly augmented NGFR expression (four- to fivefold higher than individual knockdowns) (Figure 6A–B), suggesting that their effects are synergistic.

Figure 6 with 1 supplement see all

Download asset Open asset

Given these results, a feasible possibility is that a hnRNP K/L-circZNF827 complex could facilitate transcriptional repression of NGFR by interacting with gene-regulatory regions, consistent with NGFR upregulation upon circZNF827 knockdown. To this end, publicly available ChIP-seq data (ENCODE consortium) in K562 and HepG2 cells demonstrate that hnRNP K indeed interacts with transcription regulatory regions (promoter proximal) of the NGFR gene (Figure 6—figure supplement 1C). To determine the circZNF827-dependence of a hnRNP K-containing complex that docks at the promoter region of NGFR gene in L-AN-5 cells, we next performed hnRNP K ChIP in the presence or absence of circZNF827 and assayed for the NGFR promoter region by qPCR. Our results show that hnRNP K engagement at the NGFR promoter is decreased upon circZNF827 knockdown compared to the GAPDH gene (Figure 6C), which displayed constant transcription rates in our previous BrU pulse labeling assay. We next wondered how this circRNP complex can interact with chromatin. It was recently demonstrated that hnRNP K partakes in a complex with chromatin-bound KRAB-domain zinc finger proteins (KRAB-ZNFs), and that hnRNP K is necessary for recruitment of a transcription inhibitory SETDB1/KAP1 complex, which catalyzes H3K27 trimethylation and heterochromatin formation (Thompson et al., 2015). We therefore hypothesized that ZNF827 protein, which does not harbor a discernible KRAB domain, could interact with either hnRNP K and/or its encoded circRNA and perhaps link this transcription repressive complex to the NGFR promoter. To test this hypothesis, we first conducted a ZNF827 ChIP experiment and probed for an interaction with the NGFR promoter region in the presence or absence of circZNF827. Indeed, our results suggest that ZNF827 interacts with the NGFR promoter and that the signal was significantly diminished upon circZNF827 knockdown (Figure 6D). Interestingly, we observed a similar reduction in signal at the RARA promoter region, which we had previously found to be moderately, yet significantly, upregulated (Figure 6—figure supplement 1D). To address whether ZNF827 and hnRNP K interact, we next performed ZNF827 immunoprecipitation and found it to strongly associate with hnRNP K and to a lesser extent with hnRNP L in nucleoplasmic extracts (Figure 6E, left). Upon sonication of the remainder from the Triton X-100 extracted cleared lysates (chromatin enriched), we observed an even stronger association of hnRNP K with ZNF827, further suggesting that the complex is chromatin bound (Figure 6E, right). When assessing the ability of ZNF827 to interact with circZNF827, we observed a strong enrichment over IgG (~18 fold), and ZNF827 protein co-immunoprecipitated circZNF827 more efficiently than GAPDH mRNA (~19 fold more enriched) (Figure 6F). Finally, we performed ZNF827 knockdown to test whether this would also augment expression of NGFR as observed upon hnRNP K/- L knockdown. Indeed, we observed a strong upregulation of NGFR upon ZNF827 knockdown, an effect that was further augmented in a circZNF827 knockdown background (Figure 6G–H and Figure 6—figure supplement 1E).

Taken together, our results are consistent with a model where circZNF827 represses NGFR transcription (and likely many other genes, including RARA) by bridging a hnRNP K/L-contaning inhibitory complex with their genomic loci, facilitated by the ZNF827 protein.

Mechanistically, an important outstanding question is whether circZNF827 nucleates the hnRNP-ZNF827 complex to potentially prepare/activate it for engagement with chromatin, or whether circZNF827 itself, is part of the chromatin-bound complex. To address this question, we first cloned a circZNF827 expression construct, which includes a sequence encoding 2 X MS2 hairpins (Figure 7A) and established stable inducible HEK293 cells (HEK293-circZNF827-MS2). Next, we transfected these cells, HEK293 control cells (Empty) or HEK293-circZNF827-WT (lacking MS2 hairpins) with a plasmid encoding FLAG-tagged MS2 coat protein (FLAG-MS2cp) and subsequently performed anti-FLAG ChIP analysis, to test whether the FLAG-tagged MS2cp becomes crosslinked to the NGFR promoter via circZNF287-2XMS2. The NGFR promoter signal was significantly higher in HEK293 cells harboring the circZNF827-2XMS2 expression cassette, when compared to both control cell lines and background signal on the GAPDH promoter remained constant in all three cell types (Figure 7B). Importantly, RIP experiments demonstrated that both hnRNP K and -L interact with circZNF827-2XMS2 with similar affinities as circZNF827-WT (Figure 7—figure supplement 1). These results demonstrate that circZNF827 engages the NGFR promoter, suggesting that the circular RNA nucleates the hnRNP-ZNF827-containing complex on chromatin to limit NGFR expression (Figure 7C), which in turn contributes to an important balance between neuronal differentiation and self-renewal/proliferation.

Figure 7 with 1 supplement see all

Download asset Open asset

To create plasmids for expression of dishRNAs, sense and antisense oligonucleotides were annealed and cloned into BglII/XhoI-digested pFRT/U6, resulting in vectors designated pFRT/U6-dishRNA (Kaadt et al., 2019). Subsequently, the U6-dishRNA expression cassettes were PCR-amplified from pFRT/U6-dishRNA vectors and inserted into ClaI/BsiWI-digested pCCL/PGK-eGFP-MCS (Kaadt et al., 2019). The resulting lentiviral transfer vectors were designated pCCL/U6-dishRNA-PGK-eGFP-MCS. As a negative control, an irrelevant dishRNA based on a previously described irrelevant shRNA (Jakobsen et al., 2009), which does not match any known sequence in the human genome was used throughout the study.

To generate plasmids for in vitro transcription of circRNAs, the exons encoding circTULP4, circZNF827, circHDGFRP3, circZNF609, and circSLC8A1 were PCR-amplified from cDNA prepared from RNA isolated from L-AN-5 cells. PCR-amplicons encoding circTULP4, circZNF827, circHDGFRP3, and circSLC8A1 were inserted in BamHI/NotI-digested pcDNA3/PL whereas PCR-amplicons encoding circZNF609 were inserted in HindIII/NotI-digested pcDNA3/PL. The resulting plasmids were designated pcDNA3/circRNA. The plasmid for expression of the CTC lincRNA was constructed as previously described in Seitz et al., 2017.

For exogenous expression of circZNF827, the exons encoding circZNF827 and circZNF827-hnRNPK/L mut were PCR-amplified from cDNA prepared from RNA isolated from L-AN-5 cells and inserted into PacI/SacII-digested pcDNA3.1(+)-Laccase2-MCS-exon-vector (Kramer et al., 2015). Subsequently, the Laccase-circZNF827 and Laccase2-circZNF827-hnRNPK/L mut expression cassettes was inserted into HindIII/NotI- or BamHI/XhoI-digested pcDNA5_FRT/TO resulting in vectors designated pcDNA5_FRT/TO-Laccase2-circZNF827 and -circZNF827-hnRNPK/L mut. pcDNA5-Laccase-circZNF827-2XMS2 was created by subcloning a 2xMS2 hairpin sequence (annealed phosphorylated DNA oligos) into pcDNA3.1(+)-Laccase2-circZNF827 using the unique BspE1 site, Supplementary file 2c. Positive clones were verified by sequencing and a HindIII-NotI fragment from this vector was used to insert into the HindIII-NotI sites of pcDNA5-FRT-TO. This construct was used to create stable inducible HEK293-Laccase2-circZNF827-2XMS2 cells.

pcDNA5-FLAG-MS2-coat-Twin-Streptag was created by inserting the FLAG-MS2 coat protein open-reading frame (HindIII fragment) from pNMS2-FLAG (Clement et al., 2011) into pcDNA5-Twin-Streptag. pcDNA5-Twin-Streptag was made by inserting a PCR product (containing a Twin-Streptag followed by a TEV protease site) using a pDSG-IBA-Twin-Strep-Tag vector as template (IBA-lifesciences) into the HindIII-ApaI site of pcDNA5-FRT-TO (Invitrogen).

To create plasmids for expression of FLAG-tagged RNA binding proteins (RBPs), the coding sequences of RBPs hnRNPK, hnRNPL, hnRNPU, DDX3X, and DHX9 were PCR-amplified from cDNA prepared from RNA isolated from L-AN-5 cells whereas eGFP was sub-cloned from pNEGFP (HindIII/HindIII) and inserted into either KpnI/NotI-, BamHI/NotI- or HindIII-digested pcDNA5_FRT/TO-FLAG. The resulting plasmids were designated pcDNA5_FRT/TO-FLAG-RBP. All plasmids were verified by Sanger sequencing.

All used cell lines have been periodically tested for mycoplasma contamination (PCR-based test) with negative results. L-AN-5 cells (obtained from Childrens Oncology Group - COG Cell Line and Xenograft Repository (www.cogcell.org)) were maintained in RPMI whereas SH-SY5Y (obtained from ATCC; ATCC CRL-2266) and HEK293S-Flp-In T-Rex (purchased from Invitrogen) cells were maintained in DMEM medium (Gibco, Dublin, Ireland, 32430100). For all three cell lines, the cell culture medium was supplemented with 10% fetal bovine serum (Gibco, 10082139) and 1% penicillin/streptomycin (Gibco, 15140122). P19 cells (obtained from ATCC; ATCC CRL-1825) were maintained in MEMα supplemented with 7.5% newborn calf serum (Gibco, 26010074), 2.5% fetal bovine serum (Gibco, 10082139), and 1% penicillin/streptomycin (Gibco, 15140122). HEK293 Flp-In T-Rex cells were maintained in DMEM supplemented with 10% tetracyclin-free fetal bovine serum (Gibco, 10082139) and 1% penicillin/streptomycin (Gibco, 15140122). All cells were cultured at 37°C in 5% (v/v) CO₂.

For neuronal differentiation of the neuroblastoma cell lines L-AN-5, SH-SY5Y, and P19 10 μM retinoic acid (RA) (Sigma-Aldrich, St. Louis, Missouri, United States) was added to the cell culture medium for 4 days.

The cell line with stable expression of circZNF827 was generated as previously described (Hollensen et al., 2018). Briefly, HEK293 Flp-In T-Rex cells were co-transfected with pcDNA5-FRT/TO-laccase2-circZNF827, -circZNF827-MS2, or -circZNF827-hnRNPK/L mut and a plasmid for expression of the Flp recombinase (pOG44). Cell culture medium supplemented with 100 ng/ml Hygromycin (Thermo Scientific, Waltham, Massachusetts, United States) and 10 ng/ml Basticidin S (Thermo Scientific) was used for selection of positive clones. The resulting cell line was designated HEK293 Flp-In T-Rex-circZNF827, circZNF827-MS2, and -circZNF827-hnRNPK/L mut. Tetracycline (Tet) concentrations used for titration of circRNA induction (Northern blot) were 5 ng/ml, 25 ng/ml, 100 ng/ml, and 250 ng/ml, respectively. 25 ng/ml Tet was used for RIP experiments and 250 ng/ml Tet for hnRNP K/L immunofluorescence assays.

E14 mESCs (ES-E14TGs2a; obtained from ATCC; ATCC CRL-1821) were grown on 0.1% gelatin-coated plates in 2i medium (Ying et al., 2008) containing: DMEM/F12 (Gibco, 31331) and Neurobasal (Gibco, 12348) 1:1, N2 supplement (Gibco, 17502048), B27 supplement (Gibco, 17504044), 1X glutaMax (Gibco, 35050061), 1X penicilin/streptomycin (Gibco, 15140122), 1 mM sodium pyruvate (Gibco, 11360070), 50 nM 2-mercaptoethanol (Gibco, 31350010), nonessential amino acids (Gibco, 11140076), LIF, 3 μM GSK3 inhibitor (CHIR-99021) and 1 μM MEK inhibitor (PD0325901). They were differentiated into neurons as previously described (Bibel et al., 2007) with some modifications. 4 million cells were differentiated into embryoid bodies in suspension in petri dishes for bacterial culture in 15 ml medium containing the same as before, but with 10% FBS and without LIF or GSK3 and MEK inhibitors. Every second day, the medium was changed and the embryoid bodies transferred to fresh petri dishes. On days 4 and 6, 5 μM ATRA (Sigma-Aldrich, R2625) was added to the medium. On day 8 of differentiation, the embryoid bodies were disgregated with 5% trypsin (Gibco, 15400054) and the cells plated in poly-DL-ornithine (Sigma-Aldrich, P8638) and laminin (Sigma-Aldrich, L2020) coated plates in N2 medium, containing DMEM/F12 and neurobasal 1:1, N2 supplement, sodium pyruvate, glutaMax, 15 nM 2-mercaptoethanol, and 50 μg/ml BSA. The medium was changed after 2 hr and after 24 hr. 48 hr after plating the neuronal precursors, the medium was changed to complete medium, containing B27 supplement, in addition to the N2 medium. Neurons were harvested 2 and 8 days after plating. Our E14 cell line has periodically been tested for mycoplasma contamination (PCR-based test) with negative results.

20 µg RNA from each sample was depleted of rRNA using a Ribo-Zero rRNA Magnetic Kit (Epicentre, St Louis, Missouri, United States) including the optional RiboGuard RNase inhibitor according to the manufacturer’s protocol. The concentration was normalized so that each sample contained the same amount of RNA. To 1/3 of the sample 1/10 of the recommended amount of spike-in (ERCC RNA spike-in mix, Ambion) was added, ethanol precipitated, and resuspended in ‘Elute, fragment, finish mix’ (Illumina, San Diego, California, United States). The remaining 2/3 of the sample was ethanol precipitated and resuspended in 15 µl nuclease free water. The sample was heated to 70 °C for 1 min and incubated on ice for 2 min. 5 µl RNase R mixture (Epicentre) was added to the sample before incubation at 37°C for 30 min. RNase R was removed by phenol/chloroform extraction. The RNA was resuspended in ‘Elute, fragment, finish mix’ (Illumina). Sequencing libraries were prepared using Truseq stranded RNA LT kit (Illumina) from both Ribo-Zero and Ribo-Zero/RNase R samples, by fragmentation, 1st and 2nd strand cDNA synthesis, 3′-end adenylation, ligation of adaptors, and enrichment of DNA fragments using the manufacturer’s protocol. The quality of the library was validated using an Agilent Bioanalyzer 1000 (Agilent Technologies, Santa Clara, California, United States). The samples were sequenced using the Illumina HiSeq 2500 platform with 100 bp paired-end reads (AROS Applied Biotechnology, Aarhus, Denmark).

Reads were mapped onto the mm10 genome, and circRNAs were detected and quantified using find_circ (Memczak et al., 2013), CIRCexplorer2 (Zhang et al., 2016) (v2.3.3), and ciri2 (Gao et al., 2018) (v2.0.6) using default settings except for find_circ, where a stringent mapq threshold of 40 was used for both adaptor sequences as proposed previously (Hansen, 2018). The prediction-output from all pipelines was merged and intersected, and only circRNAs detected by all three pipelines and with three-fold enrichment of backsplice-spanning reads in the RNAseR-treated samples were defined as bona fide. Expression, based on untreated samples quantified ciri2, was RPM normalized and the top100 expressed bona fide circRNAs across all samples were subjected to kmean clustering using five centers based on within-clusters sum of squared. Annotated genes (UCSC annotation) with at least one splice site in common with circRNAs were denoted as host genes, and based on host-gene annotation, exon numbers and flanking intron lengths were extracted.

The circ-to-linear ratios were based on the backsplice junction spanning reads and the mean of upstream and downstream linear spliced reads as quantified by find_circ.

To compare with human expression profiles, the top100 expressed circRNAs were converted from mm10 to hg19 coordinates using liftOver (UCSC), and only fully matched loci were considered homologous. Data is available at the NCBI Gene Expression Omnibus: GSE157788.

RNAseq from Rybak-Wolf et al (GSE65926 Rybak-Wolf et al., 2015) was solely analyzed with find_circ using stringent settings as described above.

Third-generation lentiviral vectors were produced in HEK293T cells as previously described (Hollensen et al., 2017). One day before transfection, cells were seeded in 10 cm dishes at a density of 4 × 10⁶ cells/dish. Transfections were carried out with 3.75 μg pMD.2G, 3 μg pRSV-Rev, 13 μg pMDLg/pRRE and 13 μg lentiviral transfer vector using a standard calcium phosphate or polyethylenimine transfection protocol. Medium was changed to RPMI medium one day after transfection. Two days after transfection, viral supernatants were harvested and filtered through 0.45 μm filters (Sartorius, Göttingen, Germany). All lentiviral preparations were made in at least triplicates and pooled before determination of viral titers.

To determine viral titers of lentiviral preparations, flow cytometric measurements of eGFP expression were used as previously described (Hollensen et al., 2017). One day prior to transduction, L-AN-5 cells were seeded at a density of 5 × 10⁵ cells/well in 12-well plates. For all lentiviral preparations, transductions with 10²- and 10³-fold dilutions of virus-containing supernatants were carried out. Both viral supernatants and growth medium were supplemented with 4 μg/ml polybrene. One day after transduction, medium was changed. Five days after transduction, cells were harvested and fixated in 4% paraformaldehyde (Sigma-Aldrich). eGFP expression levels were analyzed on a CytoFLEX flow cytometer (Beckman Coulter, Brea, California, United States). Lentiviral titers were calculated based on samples with between 1% and 20% eGFP-positive cells using the formula: titer (TU/ml)=F·Cn·DF/V, where F represents the frequency of eGFP-positive cells, Cn the total number of target cells counted the day the transductions were carried out, DF the dilution factor of the virus and V the volume of transducing inoculum.

One day prior to transduction with lentiviral vectors encoding circRNA-specific dishRNAs, L-AN-5 cells were seeded at a density of 6.6 × 10⁶ cells/dish in 10 cm dishes, 2.2 × 10⁶ cells/dish in 6 cm dishes, or 0.8 × 10⁶ cells/well in 6-well plates. Transductions were carried out using equal MOIs calculated based on titers determined by flow cytometry. Both viral supernatants and growth medium were supplemented with 4 μg/ml polybrene. One day after transduction, medium was changed. Two days after transduction, differentiation was initiated by addition of 10 μM RA (Sigma-Aldrich) to the cell culture medium. The L-AN-5 cells were differentiated for 4 days.

Lentiviral transduction and RA-mediated differentiation of L-AN-5 cells were carried out as described in the section ‘circRNA knockdown and differentiation of L-AN-5 cells’. After 4 days of differentiation, the L-AN-5 cells were stimulated with NGF (200 ng/ml) (Thermo Scientific) for 30 min and subsequently harvested for RNA purification.

Lentiviral transduction and RA-mediated differentiation of L-AN-5 cells were carried out as described in the section ‘circRNA knockdown and differentiation of L-AN-5 cells’. The L-AN-5 cells were cultured in 6 cm dishes containing 6 ml cell culture medium supplemented with 10 μM RA. 4 ml cell culture medium was aspirated from each 6 cm dish and pooled from cells transduced with the same dishRNA. For one dish per dishRNA, the residual medium was aspirated and 3.5 ml of the collected medium was added. For the remaining dishes, the residual medium was aspirated and 3.5 ml of the collected medium supplemented with 2 mM BrU (ThermoFisher) was added. 1 hr after addition of BrU to the cell culture medium, the cells were washed three times in cell culture medium. 50 min after removal of the BrU-containing cell culture medium the first samples including the samples not treated with BrU were harvested. Subsequently, samples were harvested after 3, 6, and 9 hr. Total RNA was purified using 1 ml TRI Reagent (Sigma-Aldrich) according to manufacturer’s protocol. circRNA knockdown and differentiation of L-AN-5 cells were verified by RT-qPCR using total RNA as described in the section ‘Quantitative PCR’. BrU-labeled RNA was immunoprecipitated as described elsewhere (Meola et al., 2016). Briefly, BrU antibodies were conjugated to magnetic beads. 15 μl Dynabeads M-280 Sheep Anti-Mouse IgG (Invitrogen, Carlsbad, California, United States) per sample were washed twice in 1x BrU-IP buffer (20 mM Tris-HCl (pH 7.5)), 250 mM NaCl, 0.5 μg/μl BSA, 20 U/ml RiboLock (Fermentas, Waltham, Massachusetts, United States) and resuspended in 1 ml 1x BrU-IP buffer with heparin (20 mM Tris-HCl (pH 7.5), 250 mM NaCl, 1 mg/ml heparin). After 30 min of incubation at room temperature on a rotator, the beads were washed in 1x BrU-IP buffer. Subsequently, the beads were resuspended in 1 ml 1x BrU-IP buffer supplemented with 0.9 μl mouse BrdU antibody (BD Biosciences, San Jose, California, United States, clone 3D4) per sample and incubated for 1 hr at room temperature on a rotator. The beads were washed three times in 1x BrU-IP buffer and resuspended in 50 μl 1x BrU-IP buffer supplemented with 1 mM 5-BrU per sample. After 30 min of incubation at room temperature on a rotator the beads were washed three times in 1x BrU-IP buffer and resuspended in 50 μl 1x BrU-IP buffer per sample. 25 μg of total RNA was diluted to 200 μl and incubated at 80°C for 2 min. 200 μl 2x BrU-IP buffer with BSA and RiboLock (20 mM Tris-HCl (pH 7.5)), 250 mM NaCl, 1 μg/μl BSA, 80 U/ml RiboLock (Thermo Scientific) and 50 μl beads conjugated with BrdU antibodies were added to the RNA samples. After 1 hr of incubation at room temperature on a rotator, the beads were washed four times in 1x BrU-IP buffer. For elution of immunoprecipitated RNA, the beads were resuspended in 200 μl 0.1% SDS. RNA was purified by phenol/chloroform extraction, ethanol precipitation and the RNA pellets were resuspended in 10 μl nuclease free water. 2 μl of immunoprecipitated RNA was used for quantification of mRNA expression levels by RT-qPCR as described in the section ‘Quantitative PCR’ except that DNase treatment was omitted and 1 μg yeast RNA (Roche, Basel, Switzerland) was added in the cDNA reaction.

The BrU-labeling and immunoprecipitation of newly labeled RNA were carried out as for the mRNA decay assay except that the cells were harvested 45 min after addition of BrU to the cell culture medium. Furthermore, after binding of the RNA to the beads, the beads were washed once in 1x BrU-IP buffer, twice in 1x BrU-IP buffer supplemented with 0.01% Triton X-100 and twice in 1x BrU-IP buffer.

Subcellular fractionation of nuclear and cytoplasmic RNA was carried out as previously described (Hollensen et al., 2018). Briefly, cells were washed in PBS, then 800 μl PBS was added and the cells were scraped off. 100 μl of the cell solution was centrifuged at 12,000 rpm for 10 s at 4°C. Cell pellets were used for purification of total RNA using 1 ml of TRI Reagent (Sigma-Aldrich) according to manufacturer’s protocol. The remaining 700 μl of the cell solution was used for subcellular fractionation of nuclear and cytoplasmic RNA. After centrifugation at 12,000 rpm for 10 s at 4°C 300 μl lysis buffer (20 mM Tris-HCl (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.5% Igepal-630 (Nonidet P-40)) were added to the cell pellets, which were then incubated on ice for 2 min and centrifugated at 1000 g for 4 min at 4°C. Cytoplasmic RNA was purified from the supernatants using 1 ml TRI Reagent (Sigma-Aldrich) according to the manufacturer’s protocol. Pellets were washed twice in 500 μl lysis buffer, subjected to a single 5 s pulse of sonication at the lowest settings (Branson Sonifier 250) and nuclear RNA was purified using 1 ml TRI Reagent (Sigma-Aldrich) according to the manufacturer’s protocol.

Cells were washed in PBS, then 800 μl PBS were added and the cells were scraped off. 80 μl of the cell solution was centrifuged at 500 g for 5 min and 200 μl lysis buffer (1x TBS, 0.5% Igepal-630 (Nonidet P-40)) were added to the cell pellets for isolation of total protein. The remaining 720 μl of the cell solution was used for subcellular fractionation of nuclear and cytoplasmic protein. After centrifugation at 12,000 rpm for 10 s at 4°C cell 300 μl lysis buffer were added and the cell pellets, which were incubated on ice for 2 min and centrifugated at 1000 g for 4 min at 4°C. The supernatants (cytoplasmic fractions) were transferred to new tubes. Pellets (nuclear fractions) were washed twice in 500 μl lysis buffer and once in 500 μl 1x TBS and resuspended in 200 μl lysis buffer. All samples were subjected to two 5 s pulses of sonication at the lowest settings (Branson Sonifier 250) followed by centrifugation at 4000 g for 25 min at 4°C. Supernatants were transferred to new tubes containing 87% glycerol (final concentration of 10%) and concentrations were adjusted using Bio-Rad protein assay (Bio-Rad, Hercules, California, United States).

RNA was purified using TRI reagent (Thermo Scientific) according the to manufacturer’s protocol. RNA samples were treated with DNase I (Thermo Scientific) according to the manufacturer’s protocol. First-strand cDNA synthesis was carried out using the Maxima First Strand cDNA synthesis Kit for qPCR (Thermo Scientific) according to the manufacturer’s protocol. qPCR reactions were prepared using gene-specific primers (Supplementary file 2d) and Platinum SYBR Green qPCR Supermix-UDG (Thermo Scientific) according to the manufacturer’s protocol. An AriaMx Real-time PCR System (Agilent Technologies) was used for quantification of RNA levels and the X₀ method was used for calculations of relative RNA levels (Thomsen et al., 2010) normalized to either GAPDH or beta-actin (ACTB) mRNA as indicated.

Gene expression analysis of 770 neuropathology-related genes were analyzed using the nCounter Human Neuropathology Panel (NanoString Technologies, Seattle, Washington, United States) and the nCounter SPRINT Profiler (NanoString Technologies) according to manufacturer’s protocol. Data analysis was carried out in the nSolver 4.0 software (NanoString Technologies) using the default settings in the nCounter Advanced Analysis Software (NanoString Technologies).

Lentiviral transduction and RA-mediated differentiation of L-AN-5 cells were carried out as described in the section ‘circRNA knockdown and differentiation of L-AN-5 cells’. Labeling of newly synthesized DNA was carried out using Click-iT Plus EdU Alexa Flour 647 Flow Cytometry Assay Kit (Thermo Scientific) according to manufacturer’s protocol. Notably, the cell culture medium of L-AN-5 cells cultured in six-well plates was supplemented with 10 μM EdU for 1.5 hr. To stain total DNA, cells with already detected EdU were resuspended in 400 μl 1x Click-iT saponin-based permeabilization and wash reagent from the Click-iT Plus EdU Alexa Flour 647 Flow Cytometry Assay Kit (Thermo Scientific). Subsequently, RNase A was added to a final concentration of 0.2 mg/ml. After 5 min of incubation at room temperature, propidium iodide was added to a final concentration of 5 μg/ml and the cells were incubated for 30 min at room temperature. Incorporated EdU and total DNA levels were analyzed on a BD LSRFortessa flow cytometer (BD Biosciences). Data analysis was carried out in the FLOWJO software (BD Biosciences). The gating strategy is shown in Figure 2—figure supplement 3A.

Cells were scraped off, pelleted and lysed for 15 min on ice in RSB100 (10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 2.5 mM MgCl2) supplemented with 0.5% Triton X-100 and 1 pill Complete protease inhibitor cocktail (Roche). The cell lysates were subjected to two 5 s pulses of sonication at the lowest settings (Branson Sonifier 250) followed by centrifugation at 4000 g for 15 min at 4°C. Glycerol was added to the supernatants (final concentration: 10%) and protein concentrations were adjusted using Bio-Rad protein assay (Bio-Rad). The protein samples were diluted in 6x loading buffer (9.8% glycerol, 12% SDS, 375 mM Tris-HCl (pH 6.8), 0.03% bromophenol blue, 10% β-mercaptoethanol), heated at 95°C for 3 min and seprated on a Novex WedgeWell 4–12% Tris-Glycine Gel (Invitrogen). Proteins were transferred to an PVDF Transfer Membrane (Thermo Scientific) using standard procedures. The membranes were blocked in 5% skimmed milk powder in PBS for 1 hr at room temperature. The membranes were incubated at 4°C overnight with primary antibodies diluted as indicated in Supplementary file 2f in 5% skimmed milk powder in PBS. After three times wash, the membranes were incubated with goat polyclonal HRP-conjungated secondary antibodies (Dako, Glostrup, Denmark) diluted 1:20,000 in 5% skimmed milk powder in PBS. After 1 hr of incubation at room temperature, the membranes were washed three times and the bound antibodies were detected using the SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific) according to the manufacturer’s protocol and using the LI-COR Odyssey system (LI-COR Biosciences, Lincoln, Nebraska, United States).

As DNA templates for in vitro transcription, pcDNA3/circRNA vectors encoding the full-length exonic sequences of five human circRNAs were used. Biotinylated RNAs were produced from 0.5 µg linearized, and phenol/chloroform extracted template using the MEGAscript T7 Transcription Kit (Ambion, Austin, Texas, United States), according to the manufacturer’s protocol with addition of 0.75 mM Biotin-14-CTP (Invitrogen) to the transcription reaction. In controls, nuclease free water was added instead of Biotin-14-CTP. The transcribed RNA was purified by phenol/chloroform extraction and dissolved in nuclease free water.

For each pull-down, 125 µL (bead volume) Pierce Streptavidin magnetic beads (Thermo Scientific) pre-washed in NET-2 buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100) were incubated with 30 µg in vitro synthesized circRNAs or 30 µg control RNA in 500 µL NET-2 buffer for 1 hr. 4°C mixing end-over-end. The conjugated beads were washed once in NET-2 buffer and incubated with 1.5 mL cell lysate prepared as follows: For each pull down, one 90% confluent 150 mm plate of differentiated L-AN-5 cells was washed in 10 mL ice cold PBS, and subjected to cell lysis in 1.5 mL hypotonic gentle lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2 mM EDTA pH 8.0, 0.1% Triton X-100) supplemented with Complete protease inhibitor cocktail (Roche, 1 pastel per 10 mL lysis buffer) for 5 min. on ice. Cells were collected by scraping and re-suspension, then supplemented with NaCl to 150 mM final concentration and incubated on ice for 5 min. Cleared cell lysate was obtained by centrifugation at 14,000 rpm, 4°C, 10 min. and supplemented with 10 µL Ribolock RNase Inhibitor (40 U/µL, Thermo Scientific) per 10 mL lysis before incubation with circRNA-coupled streptavidin beads for 1.5 hr, 4°C mixing end-over-end. From the cleared lysate 1% was mixed 1:1 with 2xSDS-load buffer (20% glycerol, 4% SDS, 100 mM Tris-HCL pH 6.8, 0.05% Bromophenol blue/Xylene cyanol and 10% β-mercaptoethanol) and kept as input sample. Following capture of proteins, beads were washed four times in NET-2 buffer and bound proteins were eluted in 40 µL preheated 2x SDS-load buffer by boiling at 90°C for 5 min. Eluates were subjected to SDS-PAGE electrophoresis and run either completely through and stained with SilverQuest Silver staining kit (Life Technologies, Carlsbad, California, United States) according to the manufacturer’s protocol, or only 1.5 cm into the gel for subsequent staining with GelCode Blue Stain Reagent (Thermo Scientific) according to the manufacturer’s protocol and excision of the bands for mass spectrometry application (see below).

Interacting proteins were identified and quantified according to previously described methods (Britze et al., 2014). Briefly, each gel lane was cut into 1 × 1 mm pieces and cysteine residues were blocked by reduction and alkylation using tris(2-carboxyethyl)phosphine and iodoacetamide, respectively. In-gel digestion was performed using trypsin and resulting peptides were extracted from gel pieces using acetonitrile and trifluoroacetic acid and finally purified on PepClean C-18 Spin columns (Thermo Scientific). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on an EASY nanoLC coupled to a Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific). Peptide samples were separated on a C-18 reverse phase column (EASY-Spray PepMap from Thermo Scientific with 25 cm length, 75 μm inner diameter, and 2 μm particle size) and eluted by a 90 min linear gradient of acetonitrile (4–40%) containing 0.1% formic acid. The MS was operated in data dependent mode, automatically switching between MS and MS2 acquisition, with mass resolution of 70,000 and 17,500, respectively. Up to 10 most intense ions were fragmented per every full MS scan, by higher energy collisional dissociation. Dynamic exclusion of 10 s was applied and ions with single charge or unassigned charge states were excluded from fragmentation.

MaxQuant software version 1.5.2.8 was applied for protein identification and label-free quantification by means of peptide peak areas (Cox and Mann, 2008). MS raw files were searched against a database consisting of 20,197 Homo sapiens sequences downloaded from Uniprot.org, August 2015. Carbamidomethylation of cysteines was set as a fixed modification, whereas methionine oxidation and protein N-terminal acetylation were set as dynamic modifications. The false discovery rate (FDR) was assessed by searching against a reverse decoy database, and FDR thresholds of protein and peptide identification were both set to 0.01.

For indirect immunofluorescence experiments, 1 × 10⁵ HEK293 Flp-In T-Rex or circZNF827_HEK293 Flp-In T-Rex cells were grown directly on poly-L-lysine coated coverslips in 12-well plates. Transcription of circRNA transgene was induced by addition of 10–250 ng/ml tetracycline and the induction profile was tested by Northern blotting in a parallel experiment. 24 hr later cells were fixed in 4% paraformaldehyde for 15 min, and permeabilized and blocked with PBS/1% goat serum (or horse serum)/0.5% Triton X-100 for 20 min. Cells were then incubated for 1–16 hr with mouse anti-hnRNPK (Abcam, Cambridge, United Kingdom), Rabbit anti-hnRNPU (Santa Cruz Biotechnologies, Dallas, Texas, United States) or mouse anti-hnRNPL (Abcam). Antibodies were used at 1:1000 dilutions. Following removal of the primary antibody, cells were incubated for 1 hr with 4 μg/mL secondary anti-IgG antibodies labeled with Alexa-594 and Alexa-488 (Molecular Probes, Eugene, Oregon, United States).

For RNA immunoprecipitation (IP) and co-immunoprecipitation (co-IP) of proteins L-AN-5 cells were seeded at a density of 6.6 × 10⁶ cells/dish in 10 cm dishes and differentiated as described in the section ‘Cell culturing’. HEK293 Flp-In T-Rex cells and HEK293 Flp-In T-Rex circZNF827 cells were seeded a density of 6.6 × 10⁶ cells/dish in 10 cm dishes. HEK293 Flp-In T-Rex and HEK293 Flp-In T-Rex circZNF827 cells were transfected with 5 μg pcDNA5/FRT-TO-FLAG-RBP and 25 μl polyethylenimine (PEI) (1 μg/μl) according to a standard PEI transfection protocol. 6 hr after transfection, RBP and circRNA expression were induced by addition of 100 ng/ml tetracycline to the cell culture medium. For IP of endogenously expressed proteins, antibodies were conjugated to Protein G dynabeads (Thermo Scientific) prior to harvest of the cells. 25 μl beads per sample were washed three times in 1 ml NET-2 buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Triton-X100). Subsequently, the beads were resuspended in 800 μl NET-2 buffer per sample and added 10 μl hnRNP K, hnRNP L, or IgG antibody per sample. After conjugation for 120 min at 4°C on a rotator, the beads were washed twice in NET-2 buffer and resuspended in 50 μl NET-2 buffer per sample. For IP of FLAG-tagged proteins, 50 μl anti-FLAG-M2 agarose slurry was washed twice in 1.5 ml NET-2 buffer and resuspended in 50 μl NET-2 buffer per sample. The cells were lysed after a single wash in PBS by addition of 1 ml ice-cold hypotonic gentle lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 1 mM EDTA, 0.25% Triton-X100, and one pill Complete protease inhibitor cocktail (Roche) per 10 ml), scraped off, and transferred to an Eppendorf tube. After incubation for 5 min on ice, 35 μl 4 M NaCl (final 150 mM) was added and the samples were incubated for 2 min on ice. The lysates were subjected to a single 5 s pulse of sonication at the lowest settings (Branson Sonifier 250) and centrifuged at 13,000 rpm for 15 min at 4°C. For input protein and RNA controls, 50 μl and 100 μl of the lysate were resuspended in 50 μl 2xSDS-load buffer (20% glycerol, 4% SDS, 100 mM Tris-HCL pH 6.8, 0.05% Bromophenol blue/Xylene cyanol and 10% β-mercaptoethanol) and 1 ml TRI Reagent (Sigma-Aldrich), respectively. The input protein control samples were incubated for 3 min at 80–90°C before storage at −20°C. The remainder of the supernatants was transferred to tubes containing 50 μl bead slurry and nutated at 4°C for 2 hr. Subsequently, the beads were washed seven times in 1.5 ml ice-cold NET-2 (5 min per wash) and protein was eluted form one third of the beads by addition of 100 μl 2xSDS-load buffer followed by incubation for 3 min at 80–90°C, whereas RNA was eluted from two thirds of the beads by addition of 1 ml TRI Reagent (Sigma-Aldrich).

Northern blots were carried out as previously described in Damgaard and Lykke-Andersen, 2011. Briefly, 10 μg RNA was separated in a 1.2% formaldehyde-agarose gel. Subsequently, the RNA was transferred to a Hybond membrane (GE Healthcare, Chicago, Illinoise, United States). The membrane was hybridized with circZNF827- or ACTB-specific [32P]-end-labeled oligonucleotides (sequences are specified in Supplementary file 2e) overnight and subsequently exposed on phosphorimager screens and visualized on a Typhoon FLA 9500 (GE Healthcare).

Lentiviral transduction and RA-mediated differentiation of L-AN-5 cells were carried out as described in the section ‘circRNA knockdown and differentiation of L-AN-5 cells’. HEK293 Flp-In T-Rex, HEK293 Flp-In T-Rex circZNF827, and HEK293 Flp-In T-Rex circZNF827-MS2 cells were seeded at a density of 5 × 10⁶ cells/dish in 10 cm dishes. Transfections were carried out with 5 μg pcDNA5-FLAG-MS2-coat-Twin-Streptag encoding the MS2 coat protein and 25 μl PEI (1 μg/μl) according to a standard PEI transfection protocol. 5 hr after transfection, MS2 coat protein and circRNA expression were induced by addition of 100 ng/ml tetracyclin to the cell culture medium. The ChIP assay including crosslinking and harvest of cells were carried out using the Pierce Magnetic ChIP Kit (Thermo Scientific) according to the manufacturer’s protocol except that sonication was carried out on a Covaris S2 ultrasonicator (settings: burst: 15%, cycles: 200, intensity: 6, cycle time: 20 min, frequency sweeping: on, de-gas: on) and 10 μl Anti-FLAG M2 beads (Sigma-Aldrich) blocked with 10 ng/ml FLAG-peptid (Sigma-Aldrich) were used for IP of the FLAG-tagged MS2 coat protein. The antibodies used for the ChIP assay are listed in Supplementary file 2f. DNA fragments were quantified as described in the section ‘Quantitative PCR’ using the gene-specific primers listed in Supplementary file 2d.

In biochemical assays (conducted in at least biological triplicates), the significance of difference between samples were calculated by a two-tailed Student’s t test to test the null hypothesis of no difference between the two compared groups. The assumption of equal variances was tested by an F test. p<0.05 was considered statistically significant. Data are presented as mean ± SD.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Proliferation assays using flow cytometry was performed at the FACS Core Facility, Aarhus University, Denmark. Karina Hjorth and Maria Vad Jakobsen are thanked for excellent technical assistance. Thanks to Serafin Pinol-Roma for sharing anti-hnRNP C1/C2 antibody.

© 2020, Hollensen et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.