A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival

摘要

class="head no_bottom_margin" id="sec1title">IntroductionFunctions of many lncRNAs, >200-nt-long transcripts lacking functional open reading frames (ORFs), depend on recruitment of multiple copies of specific RNA-binding proteins (RBPs) to repeated cis-elements (, ). For example, a decoy long noncoding RNA (lncRNA) called NORAD contains at least 17 binding sites for the RBP Pumilio (, ). Another lncRNA, Firre, may control nuclear architecture through repeat-mediated interaction with the nuclear matrix protein hnRNP U ().Several lncRNAs function as scaffolds facilitating biogenesis of non-membrane-bound cellular compartments (, , ). A classic example is the ribosome-producing organelle nucleolus that requires transcription of the 47S/45S rRNA precursors (pre-rRNA) by the RNA polymerase I for its assembly (). Other structural lncRNAs include NEAT1/MEN-epsilon/beta nucleating paraspeckles, stress-induced Sat-III transcripts involved in nuclear stress body assembly and Hsr-omega RNAs is required to form omega speckles (, , ). Interestingly, the Sat-III and the Hsr-omega RNAs contain 160- to 280-nt-long tandem repeats that may engage in multivalent interactions with corresponding RBPs (, ).Perhaps the most compelling example of multivalent recruitment of RBPs to RNA is provided by aberrant transcripts expressed in the context of neurodegenerative and neuromuscular disorders and containing genetically expanded short tandem repeats (STRs), head-to-tail concatemers of 2- to 12-nt sequence units (, href="#bib48" rid="bib48" class=" bibr popnode">Morriss and Cooper, 2017). For instance, pre-mRNAs containing expanded (CUG)n and (CCUG)n sequences contribute to pathogenesis of myotonic dystrophy by sequestering the RBP Muscleblind (MBNL1) in nuclear foci and inhibiting its splicing regulation function (href="#bib22" rid="bib22" class=" bibr popnode">Goodwin and Swanson, 2014, href="#bib48" rid="bib48" class=" bibr popnode">Morriss and Cooper, 2017).STRs occupy >3% of the reference human genome (href="#bib19" rid="bib19" class=" bibr popnode">Ellegren, 2004). However, with a notable exception of the subtelomeric repeat-containing lncRNA TERRA, the overall expression status of endogenously encoded STRs and possible biological functions of the corresponding transcripts remain poorly understood (href="#bib2" rid="bib2" class=" bibr popnode">Azzalin and Lingner, 2015, href="#bib6" rid="bib6" class=" bibr popnode">Biscotti et al., 2015). Moreover, it is likely that STR-containing RNAs are underrepresented in the existing transcriptome annotations because of the inherent difficulty in distinguishing such sequences from their close homologs, especially in the context of RNA sequencing (RNA-seq) experiments.LncRNAs and RBPs are frequently deregulated in cancer (href="#bib52" rid="bib52" class=" bibr popnode">Pereira et al., 2017, href="#bib62" rid="bib62" class=" bibr popnode">Schmitt and Chang, 2016). For example, polypyrimidine tract-binding protein (PTBP1/PTB/hnRNP I), an RBP regulating pre-mRNA processing in the nucleus and mRNA translation in the cytoplasm (href="#bib34" rid="bib34" class=" bibr popnode">Kafasla et al., 2012, href="#bib36" rid="bib36" class=" bibr popnode">Keppetipola et al., 2012), is upregulated in several types of cancer (href="#bib12" rid="bib12" class=" bibr popnode">Cheung et al., 2009, href="#bib28" rid="bib28" class=" bibr popnode">He et al., 2014, href="#bib69" rid="bib69" class=" bibr popnode">Wang et al., 2017). This has been linked with increased proliferation and invasiveness of cancer cells, as well as their ability to carry out aerobic glycolysis and evade apoptosis induced by extrinsic cues (href="#bib12" rid="bib12" class=" bibr popnode">Cheung et al., 2009, href="#bib14" rid="bib14" class=" bibr popnode">Cobbold et al., 2010, href="#bib16" rid="bib16" class=" bibr popnode">David et al., 2010, href="#bib31" rid="bib31" class=" bibr popnode">Izquierdo et al., 2005, href="#bib69" rid="bib69" class=" bibr popnode">Wang et al., 2017). PTBP1 is also a natural repressor of differentiation-specific alternative splicing events (href="#bib7" rid="bib7" class=" bibr popnode">Boutz et al., 2007, href="#bib34" rid="bib34" class=" bibr popnode">Kafasla et al., 2012, href="#bib36" rid="bib36" class=" bibr popnode">Keppetipola et al., 2012, href="#bib45" rid="bib45" class=" bibr popnode">Makeyev et al., 2007, href="#bib63" rid="bib63" class=" bibr popnode">Spellman et al., 2007, href="#bib72" rid="bib72" class=" bibr popnode">Yap et al., 2012), providing another possible explanation for its increased expression in cancer cells.However, upregulation of PTBP1 is insufficient to trigger cellular transformation on its own (href="#bib67" rid="bib67" class=" bibr popnode">Wang et al., 2008a). Possibly explaining this paradox, PTBP1 has been shown to stimulate expression of several activators of apoptosis by either altering splicing of their pre-mRNAs or increasing their translation efficiency (href="#bib5" rid="bib5" class=" bibr popnode">Bielli et al., 2014, href="#bib10" rid="bib10" class=" bibr popnode">Bushell et al., 2006, href="#bib74" rid="bib74" class=" bibr popnode">Zhang et al., 2009). How these pro-apoptotic activities are managed in transformed cells overexpressing PTBP1 is an open question. In many cancer cells, a fraction of PTBP1 is recruited to a nuclear body called perinucleolar compartment (PNC) (href="#bib21" rid="bib21" class=" bibr popnode">Ghetti et al., 1992, href="#bib46" rid="bib46" class=" bibr popnode">Matera et al., 1995, href="#bib50" rid="bib50" class=" bibr popnode">Norton and Huang, 2013), but the functional significance of this effect and the mechanisms directing PTBP1 to the PNC remain unclear.Here, we used a combination of bioinformatics and experimental approaches to uncover a number of previously unknown STR-enriched lncRNAs predicted to recruit multiple copies of cognate RBPs. An in-depth analysis of one such lncRNA reveals its critical roles in PNC assembly, regulation of PTBP1 activity, and cell survival.