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Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled retention

Karen Yap,1,4 Zhao Qin Lim,1,4 Piyush Khandelia,1,4 Brad Friedman,2,3,5 and Eugene V. Makeyev1,6 1School of Biological Sciences, Nanyang Technological University, Singapore, 637551; 2Department of Molecular and Biology, Harvard University, Cambridge, Massachusetts 02138, USA; 3The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage- specific . An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 39-terminal within these genes is repressed by the polypyrimidine tract-binding (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the and triggers their nuclear degradation. Clearance of these intron- containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the . When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of -competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context. [Keywords: coordinated expression; neuronal differentiation; presynaptic proteins; polypyrimidine tract-binding protein; intron retention; nuclear mRNA surveillance] Supplemental material is available for this article. Received January 24, 2012; revised version accepted April 30, 2012.

Higher contain multiple cell types that Nilsen and Graveley 2010). Recent studies suggest that differ in their morphological and physiological proper- ;90% of human and mouse genes may give rise to ties despite having identical or nearly identical ge- alternatively spliced transcripts, which often encode nomes. This phenotypic diversity relies on the expres- distinct protein isoforms (Pan et al. 2008; Wang et al. sion of distinct repertoires of lineage-specific genes 2008). This effectively increases genome-coding ca- coordinated in a precise spatiotemporal manner. A number pacity without expanding the number of the genes. of transcriptional and post-transcriptional mechanisms are events often occur in a tissue- known to control these elaborate gene networks (Davidson specific manner, with the largest number of unique 2006; Moore and Proudfoot 2009; Vaquerizas et al. 2009; splice forms found in the nervous system (Q Li et al. Ivey and Srivastava 2010). 2007; Pan et al. 2008; Wang et al. 2008). Alternative pre-mRNA splicing provides a remark- We and others have previously shown that the poly- able example of post-transcriptional regulation operat- pyrimidine tract-binding protein (PTBP1 in humans and ing on a genome-wide scale (Wang and Burge 2008; Ptbp1 in mice; other names: PTB and hnRNP I) func- tions as a global regulator of a neuron-specific alterna- tive splicing program (Boutz et al. 2007; Makeyev et al. 4 These authors contributed equally to this work. 2007; Spellman et al. 2007). Although this protein has 5Present address: Department of Bioinformatics and Computational Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, traditionally been considered a splicing repressor, recent USA. transcriptome-wide surveys have identified examples of 6Corresponding author. Email [email protected]. both PTBP1-repressed and PTBP1-activated alternative Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.188037.112. (Xue et al. 2009; Llorian et al. 2010). Ptbp1 tends

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Yap et al. to be expressed at high levels in nonneuronal and neural however, relatively little is known about the underlying stem cells (NSCs), whereas its expression is diminished mechanisms (Waites et al. 2005). in differentiating neurons by the nervous system-spe- cific microRNA (miRNA) miR-124 (Makeyev et al. Results 2007). Ptbp1 regulates a set of genes at the level PTBP1 also autoregulates its own expression through of mRNA abundance a negative feedback mechanism, in which an excess of this protein induces skipping of an alternative To understand the Ptbp1-controlled post-transcriptional within its own pre-mRNA and leads to the appearance network, we transfected the mouse neuroblastoma CAD of a premature termination codon (PTC) (Wollerton et al. cell line with either Ptbp1-specific (siPtbp1) or control 2004). The PTC-containing PTBP1 mRNAs are then (siControl) siRNAs and analyzed the samples by RNA rapidly degraded in the cytoplasm by the nonsense- sequencing (RNA-seq). In addition to the expected mediated decay (NMD) machinery (Isken and Maquat stimulation of the nervous system-specific alternative 2008; Nicholson and Muhlemann 2010). Using a similar splicing program (data not shown), an overall abun- NMD mechanism, Ptbp1 represses the expression of its dance of a number of transcripts either decreased (n = neuronal paralog (Ptbp2) and at least two other nervous 431; >1.5-fold; P < 0.001) (Supplemental Table S1) or system-specific genes (Gabbr1 and Dlg4) (Boutz et al. increased (n = 276; >1.5-fold; P < 0.001) (Supplemental 2007; Makeyev et al. 2007; Spellman et al. 2007; Zheng Table S2) in the Ptbp1 knockdown sample. As expected, et al. 2012). Thus, in addition to generating distinct siPtbp1 down-regulated Ptbp1 expression (;5.4-fold; protein isoforms, alternative splicing may cooperate with P = 0) (Supplemental Table S1) and up-regulated the the cytoplasmic RNA surveillance machinery to control expression of its neuron-enriched paralog, Ptbp2 levels (Lareau et al. 2007; Isken and (;3.5-fold; P = 2.1 3 10À259) (Supplemental Table S2). Maquat 2008; Barash et al. 2010). Interestingly, these large-scale transcriptome changes Several mechanisms are known to control the quality were accompanied by an increased propensity of CAD of RNAs in the nucleus (Sommer and Nehrbass 2005; cells to undergo neuron-like differentiation (Supplemen- Egecioglu and Chanfreau 2011). One such mechanism tal Fig. S1). blocks the export of unspliced pre-mRNAs from the The RNA-seq data contained a substantial number of nucleus to the cytoplasm, thus preventing their trans- intronic RNA-seq reads likely derived from the nuclear lation into aberrant polypeptides (Chang and Sharp (pre-)mRNA fraction. We reasoned that the ratio be- 1989; Legrain and Rosbash 1989). A growing body of tween intronic reads and reads originating from the evidence suggests that, in addition to correcting splic- adjacent exons—the statistic that we refer to as IRENE ing errors, nuclear mRNA surveillance may contribute (intronic reads normalized to exons)—should be a faith- to programmed changes in gene expression levels. For ful indicator of post-transcriptionally regulated genes. example, human SRSF1/ASF/SF2 protein homeostati- Strikingly, the statistically significant IRENE changes cally autoregulates its expression at least in part by induced by siPtbp1 correlated inversely with statisti- promoting the accumulation of incompletely spliced cally significant changes in the corresponding mRNA SRSF1 mRNA species in the nucleus (Sun et al. 2010). expression levels (P = 3.2 3 10À47, Fisher’s exact test) Similarly, the retention of intron 3 in the mouse (Fig. 1A, red dots). apolipoprotein E transcripts may interfere with their We focused on the subset of genes showing increased export from the nucleus to the cytoplasm (Xu et al. expression levels and reduced IRENE scores (33 genes) 2008). (Fig. 1A, top left quadrant; Supplemental Table S3). Since Regulated intron retention is widely used as a general Ptbp1 and Ptbp2 may regulate overlapping pre-mRNA strategy for coordinated expression of meiotic and sets and the loss of Ptbp1 leads to the Ptbp2 up-regulation ribosomal protein genes in budding and fission (Makeyev et al. 2007), we treated CAD cells with a mix- (Averbeck et al. 2005; Moldon et al. 2008; Cremona ture of Ptbp1- and Ptbp2-specific siRNAs (siPtbp1/2) and et al. 2010; Munding et al. 2010; Parenteau et al. 2011). repeated the RNA-seq analysis. In this case, increased However, it is currently unknown whether higher eu- mRNA expression and reduced IRENE scores were de- karyotes use similar intron-dependent strategies for tected for 102 genes (>1.5-fold changes; P < 0.001) (Sup- orchestrating their cell differentiation programs. plemental Table S4), which included 19 of the above Signal transmission from a presynaptic neuron to 33 genes. a postsynaptic cell is a major nervous system function To confirm that these effects were due to increased that depends on coexpression of hundreds of neuronal transcript abundance rather than altered splicing pat- proteins involved in signal-dependent fusion of synap- terns, we analyzed the mRNA expression levels for the tic vesicles with synaptic terminals (e.g., vesicle-spe- 31 Mouse Genome Informatics (MGI)-annotated genes cific v-SNAREs and calcium sensors and terminal- by RT-qPCR using primers specific to constitutively specific t-SNAREs), trafficking of vesicles along the spliced regions. All of these genes were significantly up- axon, and their recycling following neurotransmitter regulated upon Ptbp1 or Ptbp1/2 knockdown (Fig. 1B, release (Sudhof 2004; Wojcik and Brose 2007; DeBoer top graph; Supplemental Fig. S2A). We concluded that et al. 2008). The genes encoding these important proteins Ptbp1 and, possibly, Ptbp2 regulate the expression are tightly regulated during neuronal differentiation; levels of extensive sets of genes.

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Coordinated expression of neuronal genes

Figure 1. Ptbp1 regulates the expression levels of an extensive set of genes. (A) Relationship between changes in the IRENE values and in the corresponding mRNA expression levels following the Ptbp1 knockdown. (Red) Introns associated with a $1.5-fold change in IRENE (P < 0.001) and a simultaneous $1.5-fold change in the mRNA abundance (P < 0.001); (blue) the rest of the genes. Numbers of significantly regulated introns within specific quadrants are shown in red, and the corresponding gene numbers are shown in black. (B, top) CAD cells were transfected with Ptbp1-specific siRNA (siPtbp1), an siRNA mixture against both Ptbp1 and Ptbp2 (siPtbp1/2), or a control siRNA (siControl), and the expression levels of 31 genes from the top left quadrant in A were analyzed 72 h post-transfection by RT-qPCR. (Bottom) CAD cells were treated with CHX or DMSO (control), and the expression levels of the same 31 genes were measured using Agilent gene expression microarrays. In both analyses, data were averaged from two to three independent experiments 6SD, and the control expression levels were set to 1. (C) RNA-seq read density plots for the Stx1b, Vamp2, and Sv2a genes. Reads corresponding to the 39-terminal introns are shown in red.

Ptbp1 represses the expression of a number of genes Zc3h8) significantly increased their expression levels in in an NMD-independent manner response to CHX treatment (Fig. 1B, bottom graph; Sup- plemental Fig. S2B). Thus, the 26 remaining genes that Ptbp1 protein has previously been shown to reduce the failed to respond to the CHX treatment were likely regu- expression of Ptbp2 and Gabbr1 mRNAs through the lated by mechanisms other than NMD. NMD pathway (Makeyev et al. 2007). Since NMD is thought to function in the cytoplasm without affecting Expression levels of several NMD-independent genes nuclear (pre-)mRNA levels, Ptbp1 knockdown was positively correlate with the 39-terminal intron expected to increase the abundance of Ptbp2 and Gabbr1 splicing efficiency mRNAs and simultaneously decrease the corresponding IRENE statistics. Both genes were indeed present among (GO) analysis of the 26 NMD-independent the up-regulated genes with reduced IRENE values (Fig. genes returned ‘‘neurotransmitter transport’’ (GO:0006836) 1B, top graph; Supplemental Table S3). as the highest-scoring term (44.3-fold enrichment over To examine whether the remaining 29 genes were also the background frequency; Bonferroni-corrected P = 3.54 3 regulated by NMD, we treated CAD cells with cyclo- 10À4) (Supplemental Table S5). This corresponded to heximide (CHX), a protein synthesis inhibitor that also a subset of four neuron-specific genes encoding criti- blocks NMD-mediated mRNA degradation, and ana- cal presynaptic proteins: Stx1b (a t-SNARE), Vamp2 (a lyzed the effect of this treatment on the gene expression v-SNARE), Sv2a (a synaptic vesicle-associated regulator using an Agilent gene expression microarray. To our of Ca2+ levels), and Napb/bSNAP (a SNARE recycling surprise, only three additional genes (Dusp8, Id1,and protein) (Sudhof 2004; Wojcik and Brose 2007). Similar to

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Yap et al. the siPtbp1 effect, down-regulation of the Ptbp1 expres- readily detectable in the siControl transfected samples. sion by the miRNA miR-124 led to a significantly elevated Down-regulation of Ptbp1 or both Ptbp1 and Ptbp2 expression of these genes in CAD cells (Supplemental Fig. significantly decreased the relative abundance of the S2C; data not shown). intron-retained species (P < 4 3 10À5, single-factor Interestingly, for the Stx1b, Vamp2, and Sv2a genes, ANOVA test) (Fig. 2B,C; Supplemental Fig. S4E,F, first the most dramatic decrease in the IRENE scores follow- four Kif5a samples). Treating the cells with siPtbp2 ing Ptbp1 knockdown was observed for the 39-terminal only had no detectable effect, consistent with the low introns (Supplemental Table S3). This effect was due to background Ptbp2 expression in the presence of Ptbp1 a simultaneous decrease in the density of intronic reads (Makeyev et al. 2007). We concluded that Ptbp1 re- and an increase in the density of exonic reads (Fig. 1C). At presses splicing of the Stx1b, Vamp2, Sv2a, and Kif5a least three other protein-coding genes from the NMD- 39-terminal introns. independent list followed a similar trend: the Kif5a gene encoding a nervous system-specific kinesin heavy chain Retention of the 39-terminal introns inhibits mRNA involved in anterograde axonal transport of synaptic accumulation in the cytoplasm vesicles and other membrane-bound organelles (DeBoer et al. 2008), the Exosc2 gene encoding the Rrp4 subunit of Incompletely spliced mRNA species often fail to be the RNA exosome complex, and a nucleoporin gene, efficiently exported from the nucleus to the cytoplasm. Nup35 (Supplemental Table S3; Supplemental Fig. S2D; In line with this trend, the intron-retained forms of the data not shown). Stx1b, Vamp2, and Sv2a transcripts were up to 10 times To examine whether the increase in mRNA abundance more abundant in the nucleus than in the cytoplasm of of the NMD-independent genes led to the accumulation of untreated CAD cells (P < 0.01, t-test) (Fig. 2D,E). Nucleo- the corresponding proteins, we knocked down the Ptbp1 cytoplasmic fractionation of the siRNA-treated CAD protein in CAD cells by RNAi and analyzed the expression cells further revealed that knocking down Ptbp1 alone of Stx1b and Vamp2 proteins by immunoblotting (Fig. 2A). or both Ptbp1 and Ptbp2 dramatically reduced the per- The levels of both SNARE proteins increased noticeably in centage of the intron-retained species in the nucleus (P = the Ptbp1 knockdown sample as compared with the 5 3 10À9, single-factor ANOVA) (Supplemental Fig. siControl-treated cells (Fig. 2A). Taken together, these data S4E,F; data not shown). These treatments also appeared indicated that Ptbp1 might control the expression levels of to reduce the fraction of the intron-retained forms in the a subset of genes by modulating the 39-terminal intron cytoplasm (P = 0.04, single-factor ANOVA) (Supplemen- splicing efficiency. tal Fig. S4E,F; data not shown), although the effect was difficult to quantify accurately due to the overall low abundance of the intron-retained form in this cellular Ptbp1 represses the 39-terminal intron splicing compartment. Notably, we failed to detect Actb tran- in the Stx1b, Vamp2, Sv2a, and Kif5 pre-mRNAs scripts retaining the 39-terminal intron in the whole-cell Since the 39-terminal introns of Stx1b, Vamp2, Sv2a, and cytoplasmic samples (Supplemental Fig. S4E,F, Actb and Kif5 contained multiple consensus Ptbp1-binding panels). These species were present in the corresponding sites (Supplemental Fig. S3), we reasoned that Ptbp1 nuclear fractions, but their abundance was not affected by maydirectlyinhibitsplicing of these introns. To begin changes in the Ptbp1 and Ptbp2 expression (Supplemental addressing this hypothesis, we first determined the Fig. S4E,F, Actb panels). steady-state splicing efficiency of the four short We further assayed the effect of Ptbp1 and Ptbp2 on 39-proximal introns within the Stx1b pre-mRNA using mRNA expression levels by RT-qPCR using Gapdh RT–PCR analysis of the CAD cell nuclear fraction with mRNA as a normalization control (Fig. 2F). This anal- corresponding exon-specific primers (Supplemental Fig. ysis showed that the overall abundance of the cytoplas- S4A,B). Interestingly, the last intron (intron 9) was mic Stx1b mRNA rose more than threefold in the retained in >75% of the Stx1b transcripts, in contrast siPtbp1 and siPtbp1/2 samples as compared with the to the three upstream introns (5, 6, and 8) that were siControl-treated cells (P = 1.7 3 10À6, single-factor retained in <25% of the Stx1b transcripts (P = 4.6 3 10À8, ANOVA) (Fig. 2F). The Stx1b transcript expression also single-factor ANOVA test) (Supplemental Fig. S4A,B). increased in the corresponding nuclear fractions, albeit This difference could not be attributed simply to the 59- to a lesser extent (less than two times; P = 1.3 3 10À4, to-39 directionality of the pre-mRNA synthesis, since single-factor ANOVA) (Fig. 2F). Interestingly, the appar- thesteady-statesplicingefficiencyofthe39-terminal ent abundance of the Stx1b transcripts was sevenfold to intron of the b-actin (Actb) pre-mRNA was not signifi- eightfold higher in the nucleus than in the cytoplasm of cantly different from that of an upstream intron (Supple- the siControl- and siPtbp2-treated CAD cells (Fig. 2G). mental Fig. S4C,D). This difference decreased to approximately fourfold We then treated CAD cells with siPtbp1, siPtbp2, following the Ptbp1 or the double Ptbp1/2 knockdown siPtbp1/2, or siControl siRNAs and analyzed the Stx1b, (Fig. 2G). The regulation of the Stx1b, Vamp2, Sv2a, and Vamp2, Sv2a, and Kif5a 39-terminal intron splicing Kif5a mRNA abundance by Ptbp1 was not specific to status in the whole-cell RNA samples by RT–PCR the CAD cells, since similar results were obtained using (Fig. 2B,C; Supplemental Fig. S4E,F, first four samples). a clone of the L929 mouse fibrosarcoma cell line The 39 intron-retained forms of all four transcripts were (Supplemental Fig. S4G).

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Figure 2. Ptbp1 controls the expression of neuronal genes by repressing splicing of the 39-terminal introns. (A) Down-regulation of the Ptbp1 protein levels in CAD cells by RNAi elevates the expression of the Stx1b and Vamp2 proteins. The Ptbp1 knockdown efficiency is controlled by Ptbp1- and Ptbp2-specific antibodies. Gapdh and b-tubulin were used as lane loading controls. (B) CAD cells treated with siControl, siPtbp1, siPtbp2, or siPtbp1/2 were analyzed by RT–PCR using primer pairs designed to estimate the splicing efficiency of the 39-terminal introns. Note that both siPtbp1 and siPtbp1/2 increase the ratio between the intron-spliced (IS) and intron-retained (IR) forms. (C) Quantification of the results in B. Data are averaged from three independent experiments, 6SD. (D) Transcripts retaining the last intron are readily detectable in the nucleus but not in the cytoplasm of the CAD cell. (E) Quantification of the results in D. Data are averaged from three independent experiments, 6SD. (F) Cytoplasmic and nuclear fractions were prepared form CAD cells treated with siRNAs as in B, and the Stx1b expression levels were analyzed by RT-qPCR. (G) Quantification of the RT-qPCR data in F shows that the ratios between the nuclear and the cytoplasmic expression levels of Stx1b, Vamp2, and Sv2a transcripts reduce following Ptbp1 or Ptbp1/2 knockdown. (H,I) Splicing efficiency of a synthetic RNA substrate comprising the wild-type Stx1b exon 9–intron 9–exon 10 segment was assayed in vitro using either control-treated or PTBP1 monoclonal antibody-treated HeLa S3 nuclear extracts, and the reaction products were analyzed by either RT–PCR with a primer pair specific to exons 9 and 10 (H) or RT-qPCR with an exon junction- specific primer pair (I). As expected, no spliced products were detected at the time point 0. Note that the PTBP1 immunodepletion activates Stx1b splicing while having no effect on the splicing efficiency of the control AdV substrate. Data in I are averaged from three amplification experiments (6SD) and compared using the t-test. Splicing efficiencies in the presence of endogenous PTBP1 levels were set to 1. (J) In vitro splicing of the Stx1b and AdV substrates was assayed as in H using PTBP1-immunodepleted nuclear extracts supplemented with the indicated amounts of recombinant PTBP1. Note that the addition of PTBP1 back to the immunodepleted extracts inhibits Stx1b but not AdV splicing. (K) Data from J were averaged from three independent experiments (6SD) and compared using the t-test. Splicing efficiencies in the absence of recombinant PTBP1 were set to 1.

The above results are consistent with the model that the Ptbp1 directly inhibits splicing of the Stx1b 39-terminal Ptbp1-stimulated retention of 39-terminal introns inhibits intron the export of the incompletely spliced Stx1b, Vamp2, Sv2a, and Kif5a mRNAs from the nucleus to the cytoplasm and To test whether Ptbp1 repressed the 39-terminal intron eventually leads to their nuclear degradation. splicing directly, we synthesized a transcript comprising

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Yap et al. the Stx1b exon 9, intron 9, and exon 10 sequences and genome using an RMCE protocol modified for bacterial assayed splicing of this substrate in vitro followed by RT– artificial (BAC)-encoded transgenes (Supple- PCR and RT-qPCR analyses (Fig. 2H–K). Splicing of this mental Fig. S7A,B; K Yap, ZQ Lim, and EV Makeyev, in RNA was fairly inefficient in a control-treated HeLa S3 prep.). To distinguish between the recombinant and en- nuclear extract but was dramatically activated by immu- dogenous copies, we marked the 39 untranslated region nodepleting the endogenous PTBP1 protein (Fig. 2H,I; (UTR) of the transgenic Stx1b with a 68- (nt) Supplemental Fig. S5A). In contrast, PTBP1 depletion had sequence encoding an FRT site (Supplemental Fig. S7A). no effect on splicing of a control adenovirus-specific RNA Stx1b-FRT transgene expression was up-regulated follow- substrate (AdV) (Fig. 2H,I). To ensure that the stimulation ing Ptbp1 or Ptbp1/2 knockdown, similar to the endoge- of the Stx1b intron 9 splicing following PTBP1 immuno- nous Stx1b gene (Supplemental Fig. S7C). depletion was a specific effect, we added various amounts Importantly, deletion of either the entire intron 9 of purified recombinant PTBP1 to the immunodepleted (Dintron 9, 226-nt deletion) or its fragment containing splicing reactions and repeated the analysis (Fig. 2J,K). Ptbp1 consensus binding sites (DPS, 125-nt deletion) from Satisfyingly, increasing the PTBP1 concentrations effi- the Stx1b transgene (Supplemental Fig. S7A,B) com- ciently repressed splicing of the Stx1b but not AdV RNA pletely abolished the regulation of mRNA abundance in substrates. Similar results were obtained using in vitro response to siPtbp1 or siPtbp1/2 (Supplemental Fig. S7D). splicing assays with recombinant radioactive RNA sub- Notably, splicing of the wild-type transgenic intron 9 was strates (Supplemental Material; Supplemental Fig. S5B– regulated by Ptbp1 similarly to the endogenous intron 9, F). Overall, these data indicated that Ptbp1 directly re- whereas the DPS intron was spliced in a constitutive presses splicing of the Stx1b intron 9. manner (Supplemental Fig. S7E,F). As expected, no re- tention products were detected for the Dintron 9 trans- Stx1b intron 9 in its natural context is sufficient gene (Supplemental Fig. S7E). for the regulation A more detailed bioinformatic analysis of the mouse To identify cis-elements involved in the regulation of the Stx1b intron 9 sequence revealed two putative Ptbp1- intron 9 splicing and the Stx1b mRNA abundance, we binding surfaces: one highly conserved across placental turned to the minigene approach. Since transiently trans- mammals (PS1), and the other one poorly conserved (PS2) fected Stx1b constructs failed to recapitulate the regu- (Fig. 3E). To examine the significance of these elements, lation, possibly as a result of titrating out a saturable we mutated them in the minigene context. Interestingly, nuclear factor (data not shown), we took advantage of mutating PS1 and PS2 individually reduced the response the single-copy transgene knock-in technology based on of minitransgene expression to Ptbp1 or Ptbp1/2 knock- high-efficiency and low-background recombination-me- down, whereas the mutPS1/mutPS2 double diated cassette exchange (HILO-RMCE) (Khandelia et al. abolished the regulation completely (Fig. 3F). We con- 2011). Using this approach, we generated a population of cluded that intron 9 is necessary for mRNA abundance CAD cells expressing a ‘‘minitransgene’’ cassette in which control by Ptbp1 and Ptbp2 and that both PS1 the PS2 a short intron 9-containing fragment of the Stx1b gene was elements are likely required for optimal regulation. placed under the control of a doxycycline (Dox)-inducible promoter element containing a recombinant constitutive Intron 9 recognition by the splicing machinery is intron (TREi) (Fig. 3A). required for nuclear retention and degradation We then knocked down the expression of Ptbp1 or/and of the incompletely spliced Stx1b mRNA Ptbp2 in the transgenic cells (Supplemental Fig. S6) and assayed the minitransgene expression under the condi- Two models could account for the repressive effect of tions in which it was either fully activated (Dox+)or intron 9 retention on mRNA export: (1) Intron-bound transcribed at a low background level (DoxÀ) (Fig. 3B–D). Ptbp1 completely inhibits the intron interaction with the RT-qPCR analysis using transgene-specific primers showed splicing machinery and simultaneously hinders the ex- that, both with and without Dox, the expression of the port of the incompletely spliced mRNA to the cytoplasm. Stx1b minitransgene was significantly up-regulated fol- (2) Alternatively, the intron is recognized by the early lowing Ptbp1 or double Ptbp1/2 knockdown as compared components of the splicing machinery (e.g., by U1 with the siControl-treated cells (Fig. 3B). RT–PCR analysis snRNP, U2AF, and/or U2 snRNP), but the subsequent of the same samples indicated that splicing of the trans- splicing and mRNA export steps are arrested in the genic intron 9 was noticeably more efficient in the Ptbp1 presence of intron-bound Ptbp1. or double Ptbp1/2 knockdown samples (Fig. 3C,D). Thus, To distinguish between these possibilities, we inacti- intron 9 within its natural context is sufficient for Ptbp1- vated the 59 splice site (59ss) within the Stx1b(MCS) dependent regulation of Stx1b mRNA expression. minitransgene intron 9 by changing the consensus GU sequence to CC and analyzed the expression of the mutated mRNA [Stx1b(mut59ss)] in CAD cells treated Polypyrimidine sequences within the Stx1b intron 9 with siControl or siPtbp1/2 siRNAs (Fig. 4A). RT–PCR are necessary for the regulation analysis confirmed that the mutation completely inac- To test whether intron 9 was necessary for Stx1b gene tivated intron splicing (Fig. 4B). Notably, in siControl regulation, we integrated an ;170-kb transgenic fragment transfected cells, overall Stx1b(mut59ss) expression containing the entire mouse Stx1b gene into the CAD cell increased 3.6-fold as compared with the Stx1b(MCS)

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Coordinated expression of neuronal genes

Figure 3. Stx1b intron 9 is sufficient and necessary for the Ptbp1-dependent regulation. (A) Integration of the Stx1b minitransgene into the CAD-A13 cell genome using RMCE. (B–D) Recombinant CAD-A13 cells containing a single copy of the Stx1b minitransgene were treated with the indicated siRNAs for 48 h, followed by 24-h incubation with or without Dox. (B) RT-qPCR analysis showing that the minitransgene expression levels increase following the Ptbp1 or the double Ptbp1/2 knockdown. (C, top) The splicing status of the transgenic intron 9 was assayed by RT–PCR. (Bottom) No PCR signal was detected when reverse transcriptase was omitted from the RT reactions. (D) Quantification of the data in C indicates that a large fraction of transgenic transcripts retain intron 9 in siControl- and siPtbp2-treated samples, whereas siPtbp1 and siPtbp1/2 dramatically stimulate the intron splicing. (E) Diagram of the exon 9–intron 9–exon 10 fragment of the mouse Stx1b gene. The phastCons plot (green) shows the probability of sequence conservation across placental mammalian species (Siepel et al. 2005). Note that a fragment of intron 9 is conserved across species. The orange rectangle marks the position of a poorly conserved (C/T)n repeat predicted by RepeatMasker (http://www.repeatmasker.org). The wild-type PS1 and PS2 sequences containing consensus Ptbp1-binding sites (underlined) and the corresponding are indicated at the bottom.(F) Recombinant CAD-A13 cells containing a single copy of either the wild-type minitransgene or minitransgenes containing the PS1 and/or PS2 mutations were treated with the indicated siRNAs for 72 h, and the expression levels of the transgenic transcripts were analyzed by RT-qPCR. Note that the individual mutations reduce the minitransgene response to the Ptbp1 or the Ptbp1/2 knockdown, whereas the PS1/PS2 double mutation completely abolishes the regulation. Data in B and F are averaged from three experiments, 6SD. minitransgene (P = 0.04, t-test) (Fig. 4C). Moreover, levels were significantly up-regulated in both the cyto- Ptbp1/2 knockdown had no effect on Stx1b(mut59ss) plasm (3.3-fold; P = 0.008, t-test) and the nucleus (1.3-fold; transcript levels, unlike Stx1b(MCS), whose expres- P = 0.04, t-test) (Fig. 4D). The apparent abundance of the sion increased 2.2-fold following this treatment (P = Stx1b(mut59ss) transcripts was comparable between the 0.03, t-test) (Fig. 4C). nuclear and cytoplasmic fractions in both siControl- and siPtbp1/2 also had little effect on the apparent abun- siPtbp1/2-treated cells (Fig. 4E). On the other hand, dance of the Stx1b(mut59ss) transcript in the cytoplasmic Stx1b(MCS) transcripts were enriched 5.6-fold in the and nuclear fractions. In contrast, Stx1b(MCS) transcript nuclei of the siControl-treated cells and 2.3-fold in the

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Figure 4. Intron 9 recognition by the splicing machinery is necessary for the Ptbp1-dependent regulation of Stx1b mRNA abundance. (A) Diagram of the Stx1b minitransgenes. (B, top) CAD-A13 cells containing the transgenes depicted in A were treated with either siControl or siPtbp1/2 for 72 h, and the total RNAs from the whole cells or the two subcellular fractions were analyzed by RT–PCR to examine the transgenic intron 9 splicing. (Bottom) No PCR products were detected in the absence of reverse transcriptase. (C) RT-qPCR analysis showing that the mutation of either 59ss or both 59ss and 39ss significantly increases the transgene expression levels in the siControl-treated cells. Moreover, unlike Stx1b(MCS), the expression of Stx1b(mut59ss) and Stx1b(mut59ss/mut39ss) minitransgenes is not activated by siPtbp1/2. (D) RT-qPCR analysis showing that siPtbp1/2 stimulates the expression levels of Stx1b(MCS) but not Stx1b(mut59ss) or Stx1b(mut59ss/mut39ss) in the nucleus and the cytoplasm. (E) Quantification of the RT-qPCR data in D suggesting that the siPtbp1/2 treatment also reduces the originally high ratio between the nuclear and cytoplasmic levels of Stx1b(MCS) but not Stx1b(mut59ss) and Stx1b(mut59ss/mut39ss) transcripts.

nuclei of siPtbp1/2-treated cells (Fig. 4E). Similar to the Strikingly, the most prominent increase in the Stx1b Stx1b(mut59ss) behavior, Stx1b(mut59ss/mut39ss) tran- mRNA expression was detected in the Tpr knockdown scripts with mutations in both the 59ss and the 39ss were samples (Fig. 5B). This was not an off-target effect, since efficiently expressed in the nuclear and the cytoplasmic an shRNA against a distinct Tpr-specific sequence stim- compartments regardless of the Ptbp1 and Ptbp2 concen- ulated Stx1b expression in a similar manner (data not trations (Fig. 4A–E). These results suggested that the shown). Stx1b expression was also stimulated by the Dis3 nuclear retention and nuclear degradation of incom- and Exosc9 knockdowns and, to a lesser extent, the pletely spliced Stx1b mRNAs require a functional in- Exosc10 and Exosc1 knockdowns (Fig. 5B). No significant teraction between intron 9 and the splicing machinery. changes in the Stx1b levels were detected in the samples expressing Xrn2- and Pabp2-specific shRNAs (Fig. 5B). Stx1b expression is regulated by the nuclear mRNA Largely similar effects were observed for the Vamp2, surveillance machinery Sv2a, and Kif5a mRNAs, except for the lack of significant effect of the Exosc9 knockdown in the case of Vamp2 and The data presented above are consistent with the model the Exosc1 knockdown in all three cases (Supplemental that incompletely spliced mRNAs encoding presynaptic Fig. S8). Importantly, the levels of the Hprt and Actb proteins are retained in the nucleus and degraded by the ‘‘housekeeping’’ mRNA controls were not significantly nuclear mRNA quality control machinery (e.g., see Fig. up-regulated in the knockdown samples (Fig. 5C; Supple- 2F). To identify factors involved in this process, we mental Fig. S8). We concluded that intron-retained RNAs knocked down several known nuclear RNA surveillance are specifically destabilized in the nucleus in a Tpr- and components, including catalytic (Exosc10/Rrp6 and Dis3/ exosome-dependent manner. Rrp44) and structural (Exosc1/Csl4 and Exosc9/Rrp45) subunits of the exosome complex; a nuclear 59-to-39 Ptbp1 expression correlates with 39-terminal exonuclease, Xrn2; a nuclear pore protein, Tpr (Mlp1/2 intron splicing and mRNA abundance homolog); and a nuclear poly(A)-binding protein, Pabp2 of neuronal genes in vivo (Pab2 homolog) (Fig. 5A; Galy et al. 2004; Houseley and Tollervey 2009; Tomecki and Dziembowski 2010; Coyle To test whether the above mechanisms functioned in the et al. 2011; Kiss and Andrulis 2011; Lemieux et al. 2011). context of development, we analyzed the expression of

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Figure 5. Nuclear RNA surveillance machinery is involved in the Stx1b regulation. (A) CAD-A13 cells encoding stably integrated shRNAs under the control of a Dox-inducible promoter (Khandelia et al. 2011) were treated with 2 mg/mL Dox for 72 h, and the knockdown efficiencies were analyzed by RT-qPCR. Shown are residual mRNA expression levels normalized to the expression of the corresponding mRNAs in the presence of a firefly luciferase-specific shRNA control. Values are averaged from three amplification experiments, 6SD. Note that in all five cases, mRNA expression is noticeably diminished by the gene-specific shRNAs. (B) Stx1b mRNA expression levels were assayed by RT-qPCR following the knockdown of the indicated components of the RNA surveillance machinery. Note that shRNA specific to the nuclear pore-associated protein Tpr and the exosome subunit Dis3 leads to statistically significant accumulation of the Stx1b mRNA. (C) None of the shRNAs tested in this experiment led to significant up-regulation of the control Hprt mRNA. In B and C, the P-values were calculated using a two-tailed t-test assuming unequal variances and shown only for the samples showing significant (P # 0.05) up-regulation of mRNA expression in response to shRNA treatment as compared with the luciferase shRNA control. Data are normalized to the expression levels of Gapdh mRNA and are averaged from at least three amplification experiments, 6SD.

the Ptbp1 protein and its SNARE targets, Stx1b and Quantitative analyses showed that the Ptbp1 expres- Vamp2, in embryonic day 13.5 (E13.5) mouse brains (Fig. sion levels correlated positively with the relative abun- 6A). Ptbp1 was expressed predominantly in the neuro- dance of the retained mRNA species in the corresponding epithelial layer containing proliferating NSCs, whereas tissues (Spearman’s correlation coefficient r = 0.8, P = 4.0 3 Stx1b and Vamp2 showed reciprocal staining patterns, 10À6) and negatively with the overall expression levels of with the maximal expression in the mantle layer contain- the Stx1b, Vamp2, Sv2a, and Kif5a genes (r = À0.83, P = ing neurons at different stages of differentiation (Fig. 6A). 7.0 3 10À7) (Fig. 6G; Supplemental Fig. S9E). These The expression of mature miR-124 miRNA in the de- results are consistent with the role of Ptbp1 as a negative veloping neural tube was reciprocal to the Ptbp1 expres- regulator of the Stx1b, Vamp2, Sv2a, and Kif5a genes in sion pattern, as reported previously (Makeyev et al. 2007), vivo. and overlapping with the Stx1b expression pattern (Sup- plemental Fig. S9A,B). Ptbp1 regulates Stx1b expression in primary We further examined 39-terminal intron splicing for the mouse cells Stx1b, Vamp2, Sv2a, and Kif5a transcripts in adult and embryonic mouse tissues (Fig. 6B–F; Supplemental Fig. As a direct test of the Ptbp1 repressor activity in primary S9C,D). Although the four transcripts were expressed in mouse cells, we knocked down Ptbp1 expression in virtually all samples (except for the lack of the Sv2a embryonic cortical NSCs by RNAi. Immunoblot analysis expression in the adult liver), the percentage of the of the siPtbp1- and siControl-treated samples suggested transcripts retaining the 39-terminal introns varied dra- that the reduction in the Ptbp1 protein levels led to matically across the tissues. Virtually no intron-retained a noticeable increase in the Stx1b protein expression products were detected in the total adult brain or its (Fig. 7A). This was accompanied by a 2.1-fold decrease nuclear and cytoplasmic fractions (Fig. 6B,C; Supplemen- in the relative abundance of the intron 9-retained Stx1b tal Fig. S9C,D). We also failed to detect intron-retained transcripts (Fig. 7B) and a modest but significant increase Vamp2, Sv2a, and Kif5a RNAs in unfractionated E12.5 in the overall Stx1b mRNA abundance (P = 6.5 3 10À5) brain samples (Fig. 6B,C; Supplemental Fig. S9). However, (Fig. 7C). The increased Stx1b expression was not due to these species were detected in the corresponding nuclear siPtbp1-induced differentiation, since virtually all cells in fractions (Fig. 6B; Supplemental Fig. S9C). Substantial the siControl- and siPtbp1-treated samples expressed the amounts of the intron 9-retained Stx1b transcripts were NSC marker nestin (Supplemental Fig. S10A). Ptbp1 expressed in both the total E12.5 brain samples and the knockdown did not change the expression of Stx1a, nuclear fraction, while being noticeably depleted from a Stx1b paralog lacking extensive Ptbp1-binding motifs the cytoplasmic fraction (Fig. 6B,C). Strikingly, all three within its last intron (Fig. 7C). Similarly, transfection of nonneural tissues contained readily detectable amounts primary mouse embryonic fibroblasts (PMEFs) with of the intron-retained transcripts (Fig. 6D,E; Supplemen- siPtbp1/2 decreased the relative abundance of the intron tal Fig. S9D). 9-retained species 10-fold (Fig. 7D) and increased the

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Figure 6. Ptbp1 expression pattern is consistent with its role as a regulator of presynaptic genes in vivo. (A) Immunofluorescence analyses of E13.5 medulla sections of the hind brain show that the Ptbp1 protein is expressed in the NSC-containing neuroepithelial layer (NL) lining the fourth ventricle (FV) but not in the mantle layer (ML) containing neurons at different stages of differentiation. Stx1b and Vamp2 proteins are expressed in a strictly reciprocal manner. The sections are additionally stained with antibodies against Map2, a marker of mature neurons. (B) Total RNAs were purified from the entire adult and E12.5 embryonic mouse brains or the corresponding cytoplasmic and nuclear fractions, and the Stx1b, Vamp2, and Sv2a 39-terminal intron splicing was analyzed by RT–PCR. Nuclear (45S pre-rRNA) and cytoplasmic (7SL) RNA markers were analyzed by RT–PCR to confirm the nucleocytoplasmic fractionation quality. (C) Quantification of the results in B.(D,E) The analyses in B and C were repeated for six adult and embryonic tissue samples. (F,G) RT-qPCR analyses of Stx1b, Vamp2, Sv2a, Kif5a, and Ptbp1 expression in vivo.

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Figure 7. Ptbp1 regulates the Stx1b expression in NSCs and nonneuronal cells. (A) Neurosphere cultures were nucleofected once with 100 pmol or twice with 50 pmol of either siControl or siPtbp1, and the expression of the Ptbp1 and Stx1b proteins was analyzed by immunoblotting. The Stx1b expression increased noticeably following the Ptbp1 knockdown. (B,C) The neurosphere cultures nucleofected once with 100 pmol of siRNAs were additionally analyzed by RT–PCR to examine the Stx1b intron 9 splicing status (B) and RT-qPCR to assess the Stx1b expression levels (C). (D,E) PMEFs were transfected with siControl or siPtbp1/2 and analyzed by RT– PCR and RT-qPCR as in B and C.(F, top) EGFP and Ptbp1/EGFP expression plasmid used in nucleofection experiments. (Bottom) A flow chart of the experiment carried out to examine the effect of Ptbp1 overexpression in primary cortical neurons. (G) RT-qPCR analysis confirming that Ptbp1/EGFP-nucleofected neurons express larger amounts of Ptbp1 mRNA than their EGFP-only counterparts. (H)RT- qPCR analysis showing that neurons overexpressing Ptbp1 express significantly less Stx1b and Vamp2 mRNA. (I) Quantification of RT– PCR analyses carried out as in B and D, demonstrating significantly elevated retention of the Stx1b- and Vamp2-regulated introns in the presence of Ptbp1. Data in C, E,andG–I are averaged from at least three independent amplification experiments (6SD) and compared using the t-test. (J) Model for coordinated regulation of presynaptic genes through Ptbp1-controlled splicing of the 39-terminal introns. expression of the Stx1b (2.1-fold, P = 1.0 3 10À4, t-test) but miR-124(Makeyevetal.2007)—would reduce presyn- not the Stx1a mRNA (Fig. 7E). aptic gene expression levels, we nucleofected primary To test whether rescue of the Ptbp1 levels in post- cortical neurons from E15.5 mouse embryos (Supplemen- mitotic neurons—where it is naturally diminished by tal Fig. S10B) with a plasmid encoding a constitutively

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Yap et al. expressed EGFP marker and a Dox-inducible Ptbp1 Tyagi and colleagues (Vargas et al. 2011). Although not cDNA lacking its natural 39 UTR to bypass miR-124 shown experimentally, we predict that regulation of regulation (Fig. 7F). Nucleofected EGFP-positive neurons intron splicing in these cases may also be used as a were then isolated from neuronal cultures by fluorescence- mechanism for regulating the abundance of the corre- activated cell sorting (FACS) and analyzed by RT-qPCR sponding mRNAs and that the mechanistic link be- and RT–PCR. Notably, steady-state levels of Stx1b and tween intron retention status, utilization of alternative Vamp2 but not Actb mRNA decreased significantly in the exons, and mRNA steady-state expression levels in presence of exogenous Ptbp1 (Fig. 7G,H). Moreover, Ptbp1 mammalian cells might be substantially more preva- overexpression arrested splicing of the regulated Stx1b and lent than currently thought. Vamp2 introns, as expected (Fig. 7I). We concluded that Of note, the Stx1b splice form retaining intron 9 has Ptbp1 represses presynaptic gene expression in vivo. previously been hypothesized to generate a truncated Stx1b protein (Pereira et al. 2008). While we do not exclude the possibility that minute amounts of this Discussion protein may indeed be produced under some circum- Intron retention is a major form of alternative splicing in stances, the significant nuclear enrichment of the intron metazoan organisms that contributes to proteome di- 9-containing Stx1b mRNA species in both neuroblastoma versity, regulates the efficiency of translation, and mod- cells and embryonic tissues argues that the main function ulates intracellular mRNA localization (Galante et al. of this intron is to regulate mRNA abundance. 2004; Denis et al. 2005; Mansilla et al. 2005; Marinescu Several lines of evidence argue that the Stx1b, Vamp2, et al. 2007; Wang and Burge 2008; Bell et al. 2010; Nilsen Sv2a,andKif5a genes are controlled by a mechanism and Graveley 2010; Buckley et al. 2011). Here we show distinct from the NMD. (1) These genes are not up- that the RNA-binding protein Ptbp1 coordinately regu- regulated by CHX (Fig. 1B). (2) The 39 position of the lates the expression levels of at least four functionally regulated introns makes NMD an unlikely scenario, since linked neuron-specific genes (Stx1b, Vamp2, Sv2a, and the 39-terminal exon–exon junction is located upstream of Kif5a) by inhibiting the 39-terminal intron splicing and a translation termination codon regardless of intron splic- thus promoting nuclear retention and nuclear degrada- ing status. (3) The Stx1b minitransgene lacking a func- tion of the incompletely spliced mRNAs (Fig. 7J). tional translation initiation codon is regulated at the This regulation mechanism adds a compelling example mRNA abundance level (Fig. 3A–D), and this regulation to the growing list of RNA-binding proteins that function is abolished by mutation of the intronic 59ss, which is not as post-transcriptional master regulators coordinating the expected to result in a PTC (Fig. 4). Moreover, trans- expression of functionally linked genes (Keene 2007). location of the intron-retained Stx1b transcripts to the There is also an interesting parallel between the Ptbp1- cytoplasm by inactivating the intronic splice sites (Fig. 4) controlled pathway uncovered in our study and the leads to a significant increase in the mRNA levels, which transcriptional repression of neuronal genes in nonneuro- rules out models invoking any form of cytoplasmic in- nal cells by the REST/NRSF complex (Ballas and Mandel stability of intron-containing transcripts. 2005). Similar to Ptbp1, components of this transcrip- The requirement for functional splice sites (Fig. 4) tional repressor complex are expressed at high level in suggests that the regulation circuitry identified in our nonneuronal cells, and reduced expression of these pro- study involves interaction of the retained introns with teins in mature neurons is essential to activate a number the pre-mRNA splicing machinery. Interestingly, the U1 of neuron-specific genes containing the REST/NRSF- snRNP and U2AF complexes interacting with the 59ss binding motifs (Ballas and Mandel 2005). and the 39ss have been shown to facilitate nuclear re- While further work will be required to address the tention of incompletely spliced transcripts (Rain and functional significance of the 39-terminal location of the Legrain 1997; Takemura et al. 2011). Nuclear retention regulated introns, at least one additional gene (Klc1) of incompletely spliced transcripts has additionally been encoding a Kif5a-interacting kinesin light chain appears shown to be modulated by cis-regulatory elements. Some to be regulated through a related mechanism. Klc1 encodes intron-containing retroviral and cellular transcripts are multiple splice forms, some of which are restricted to the known to be efficiently translocated to the cytoplasm due nervous system (DeBoer et al. 2008). Our RNA-seq data to their interaction with the Tap/NXF1 or Crm1 export indicate that the effect of Ptbp1 and, possibly, Ptbp2 on factors (Cullen 2003; Li et al. 2006). It is possible that Klc1 mRNA is threefold: (1) reduced overall abundance, (2) similar strategies are used by other incompletely spliced retention of the two 39-terminal introns flanking the mRNA species that have been shown to accumulate in neuron-specific cassette exon, and (3) repression of the the cytoplasm (Denis et al. 2005; Mansilla et al. 2005; neuron-specific penultimate cassette exon and the Marinescu et al. 2007; Buckley et al. 2011). On the other 39-terminal alternative exon (Supplemental Fig. S11). hand, specialized elements have been described that Notably, introns neighboring alternative exons regu- promote RNA retention in the nucleus (Taniguchi et al. lated by the TDP-43 and NOVA splicing regulators have 2007). Further work will be required to understand the recently been shown to change their retention status cis-regulation underlying nuclear retention of incom- during brain development (Ameur et al. 2011). This effect pletely spliced presynaptic mRNAs. might be explained by a largely post-transcriptional splic- Our RNAi experiments (Fig. 5; Supplemental Fig. S8) ing of introns adjoining repressed exons demonstrated by argue that the incompletely spliced Stx1b mRNA retained

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Coordinated expression of neuronal genes in the nucleus is cleared by a branch of the nuclear RNA CAD cells with Trizol (Invitogen), and the poly(A)+ mRNA surveillance machinery comprising the exosome com- fractions were prepared using a Dynabeads mRNA Direct kit plex and Tpr, a mammalian homolog of the nuclear (Invitogen). The purified mRNA samples were partially hydro- 2+ pore basket proteins Mlp1/2 previously implicated in lyzed for 2.5 min at 94°C in the presence of Mg , which was nuclear surveillance of unspliced mRNA in Saccharomy- followed by the first strand cDNA synthesis with the SuperScript III and random primers (Invitrogen). The second strand cDNA ces cerevisiae (Galy et al. 2004; Houseley and Tollervey synthesis and adapter ligation steps were carried out as recom- 2009; Tomecki and Dziembowski 2010; Coyle et al. 2011). mended by Illumina. DNA fragments of 350 bp 6 50 bp were then The involvement of Tpr in the surveillance of natural purified with SizeSelect E-gels (Invitrogen) and amplified by 15 intron-retained mRNA species is an important finding, cycles of PCR using Phusion DNA polymerase (New England since the role of this protein in the metabolism of in- Biolabs) and Illumina primers. Sequencing was carried out completely spliced RNAs in the mammalian system has using a Genome Analyzer IIx (Illumina). The RNA-seq data thus far been documented only for recombinant tran- were analyzed using the ExpressionPlot pipeline (Friedman and scripts containing intronic Tap/NXF1-dependent nuclear Maniatis 2011). The RNA-seq data have been deposited in export signals (Coyle et al. 2011). NCBI Gene Expression Omnibus (accession no. GSE37933). Interestingly, of the four exosome subunits analyzed in our study, knockdown of Dis3/Rrp44, Exosc10/Rrp6, and RMCE Exosc9/Rrp45 led to comparable up-regulation of the CAD-A13 cells expressing Stx1b minitransgenes and shRNAs intron-retained transcripts (except for the lack of signif- against RNA surveillance components were generated as de- icant effect of shExosc9 on Vamp2 expression), whereas scribed (Khandelia et al. 2011). Briefly, 2 3 105 CAD-A13 cells down-regulation of Exosc1/Csl4 resulted in noticeably were transfected with a mixture containing 99% of a recombina- less-pronounced effects (Fig. 5; Supplemental Fig. S8). tion donor plasmid and 1% of the Cre expression plasmid These data suggest that only a subset of exosomal com- (pEM784) using Lipofectamine 2000 (Invitrogen). Transgenic ponents may function in the nuclear surveillance of in- cells populations were obtained by treating the transfected cells completely spliced RNAs, consistent with the recently with 2.5–5 mg/mL puromycin for 7–10 d and pooling the proposed ‘‘exozyme’’ hypothesis (Kiss and Andrulis 2011). puromycin-resistant colonies. Single-copy integration of BAC- encoded FRT-marked Stx1b transgenes was carried out similarly, In conclusion, this study suggests that Ptbp1-con- except five to six individual puromycin-resistant colonies were trolled intron retention synchronizes the expression of picked up in this case and propagated independently. Clones critical presynaptic proteins during neuronal differentia- with correctly integrated Stx1b transgenes were used in the tion and counters their aberrant expression in nonneuro- subsequent experiments. A complete description of the BAC nal cells. We predict that similar mechanisms might RMCE protocol will be published elsewhere (K Yap, ZQ Lim, and contribute to the regulation of other functionally linked EV Makeyev, in prep.). gene networks in higher eukaryotes. See the Supplemental Material for additional details.

Materials and methods Acknowledgments We thank Neal Copeland, Nancy Jenkins, and Robin Reed for RNAi reagents; Rick Myers’s laboratory for the help with RNA-seq; and The parental CAD cells (Y Li et al. 2007) and their derivatives Tom Maniatis and Snezhka Oliferenko for valuable discussions. were transfected with Ptbp1- and/or Ptbp2-specific or control We also thank the NTU Protein Production Platform for the help ON-TARGETplus siRNAs (Dharmacon) using Lipofectamine with protein purification. This work was supported by the 2000 (Invitrogen) and analyzed 72 h post-transfection. Expression National Research Foundation grant NRF-RF2008-06 (to E.V.M.). of shRNAs was induced by treating corresponding CAD-A13 cell pools with 2 mg/mL Dox for 72 h as described (Khandelia et al. 2011). To knock down Ptbp1 and Ptbp2 expression in PMEF cells, References they were transfected with the corresponding siRNAs and RNAi Ameur A, Zaghlool A, Halvardson J, Wetterbom A, Gyllensten MAX (Invitrogen) twice over a 36-h interval so that the cells were U, Cavelier L, Feuk L. 2011. Total RNA sequencing reveals exposed to siRNA for 72 h in total. To deliver siRNAs into nascent and widespread co-transcriptional splic- neurosphere cultures, we used the Amaxa mouse NSC nucleo- ing in the human brain. Nat Struct Mol Biol 18: 1435–1440. 6 fection protocol with minor modifications. Briefly, 4 3 10 cells Averbeck N, Sunder S, Sample N, Wise JA, Leatherwood J. 2005. were nucleofected with 100 pmol of corresponding siRNA and Negative control contributes to an extensive program of plated onto 6-cm culture dishes coated with polyornithine and meiotic splicing in fission yeast. Mol Cell 18: 491–498. fibronectin. The cells were harvested 72 h following the nucle- Ballas N, Mandel G. 2005. The many faces of REST oversee ofection. Alternatively, we used two rounds of nucleofection, epigenetic programming of neuronal genes. Curr Opin Neu- each with 50 pmol of siRNA. In this case, the cells were robiol 15: 500–506. maintained as neurospheres for 36 h following the first round Barash Y, Calarco JA, Gao W, Pan Q, Wang X, Shai O, Blencowe and as adherent culture for 36 h following the second round of BJ, Frey BJ. 2010. Deciphering the splicing code. Nature 465: nucleofection. 53–59. Bell TJ, Miyashiro KY, Sul JY, Buckley PT, Lee MT, McCullough R, Jochems J, Kim J, Cantor CR, Parsons TD, et al. 2010. RNA-seq Intron retention facilitates splice variant diversity in cal- RNA-seq libraries were prepared in principle as described (Mortazavi cium-activated big potassium channel populations. Proc et al. 2008). Total RNAs were extracted from siRNA-treated Natl Acad Sci 107: 21152–21157.

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Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention

Karen Yap, Zhao Qin Lim, Piyush Khandelia, et al.

Genes Dev. 2012, 26: Access the most recent version at doi:10.1101/gad.188037.112

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