Multiple and Specific mRNA Processing Targets for the Major Human hnRNP Proteins

Abstract

Alternative splicing is a key mechanism regulating gene expression, and it is often used to produce antagonistic activities particularly in apoptotic genes. Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins form a family of RNA-binding proteins that coat nascent pre-mRNAs. Many but not all major hnRNP proteins have been shown to participate in splicing control. The range and specificity of hnRNP protein action remain poorly documented, even for those affecting splice site selection. We used RNA interference and a reverse transcription-PCR screening platform to examine the implications of 14 of the major hnRNP proteins in the splicing of 56 alternative splicing events in apoptotic genes. Out of this total of 784 alternative splicing reactions tested in three human cell lines, 31 responded similarly to a knockdown in at least two different cell lines. On the other hand, the impact of other hnRNP knockdowns was cell line specific. The broadest effects were obtained with hnRNP K and C, two proteins whose role in alternative splicing had not previously been firmly established. Different hnRNP proteins affected distinct sets of targets with little overlap even between closely related hnRNP proteins. Overall, our study highlights the potential contribution of all of these major hnRNP proteins in alternative splicing control and shows that the targets for individual hnRNP proteins can vary in different cellular contexts.Alternative splicing is a critical process that ensures the production of a multitude of proteins from a limited set of mammalian genes (7, 41). Alternative splicing decisions are regulated by a large collection of RNA-binding proteins (RBPs) that bind to pre-mRNAs in the nucleus (5). The heterogeneous nuclear ribonucleoparticle (hnRNP) proteins are among the most abundant of such proteins, and more than 20 of them have been characterized and given alphabetical names based on size from hnRNP A1 to hnRNP U (17). These proteins have been implicated in a variety of biological processes including telomere biogenesis, translation, and RNA stability, and several (e.g., hnRNP A1, A2, F, H, I [PTB], G, and L) have documented roles in splicing (34, 38). hnRNP A1 has been implicated in the splicing control of many genes, including the A1 gene itself, the caspase-2 gene, c-src, and the SMN2 gene (12), and several exons of human immunodeficiency virus type 1; the very similar hnRNP A2 protein (68% identity) appears to display comparable activity (4,10,29,42). While the related hnRNP F and H proteins play a role in splicing control of many genes, including c-src, Bcl-x, cystathionine ␤-synthase, and several HIV alternative exons (38), it is unclear whether F and H have completely redundant activities. hnRNP I (PTB) is another well-known splicing regulator that has been mostly associated with splicing repression (54,59). A recent global analysis of PTB and its neural paralogue nPTB has revealed their role in the control of murine neuron-specific splicing (8). hnRNP G (RBM-X) has been implicated in the splicing...

CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia

“…RNA splicing requires a complex interplay of multiple RNA-binding proteins that are equipped with domains to bind sequence motifs on single stranded RNA to ensure accurate determination of exon recognition. In silico interrogation of the wild-type pre-mRNA segment derived from the 132-bp mutation-rich intronic sequence between exons 12 and 13 for accessible splicing factor binding sites resulted in identification of multiple potential binding sites for the RNA-binding proteins hnRNP-E2/ PCBP, hnRNP-I/PTB, and hnRNP-L, three members of the heterogenous nuclear ribonucleoprotein family of splicing factors that act as global regulators of alternative splicing and are abundantly expressed in infant leukemias (40)(41)(42)(43)(44)(45)(46)(47)(48)(49) (Fig. 2A.1).…”

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

“…hnRNP A1 bound to ISS sequences can also promote exon exclusion, possibly through a mechanism that involves self-interaction of A1 molecules bound to distal sites and loop formation (Blanchette and Chabot 1999;Kashima et al 2007). While best characterized as repressors of splicing, hnRNP A/B proteins can also activate inclusion of some exons (Martinez-Contreras et al 2006;Venables et al 2008). As observed in PKM splicing regulation , hnRNP A1 and A2 frequently function redundantly in splicing regulation (Kashima and Manley 2003;Licatalosi and Darnell 2010).…”

“…As observed in PKM splicing regulation , hnRNP A1 and A2 frequently function redundantly in splicing regulation (Kashima and Manley 2003;Licatalosi and Darnell 2010). However, a genome-wide approach indicated that hnRNP A1/A2 targets may, in fact, be quite divergent (Venables et al 2008). Additional investigation is required to establish the extent of their redundancy, and whether any redundancy exists with a third family member, hnRNP A3, which shares more identity with hnRNP A1 than A2 does.…”

Proteomics of tissue factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control

“…It is worth noting that 427 alternative splicing can be regulated by hnRNP proteins binding exonic splicing silencer 428 elements, thus excluding exons from mature mRNA. One study has found that hnRNP K may 429 play a prominent role in alternative splicing control because nearly half of 56 alternative splicing 430 events in apoptotic genes were affected upon hnRNP K depletion, either enhancing or 431 suppressing exon inclusion [57]. In support of our finding, it has been shown that hnRNP K 432 represses the production of the pro-apoptotic Bcl-x S spliced isoform, whereas its down-regulation 433 enhances the splicing of Bcl-x L to Bcl-x S [58].…”