Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron 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 Cell 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 genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins 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 introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm 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 exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-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 gene 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 eukaryotes 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 Alternative splicing 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. exons (Xue et al. 2009; Llorian et al. 2010). Ptbp1 tends GENES & DEVELOPMENT 26:1209–1223 Ó 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org 1209 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press 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 exon 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 gene expression 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 yeasts (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