While all of the hnrnps are present in the nucleus, some seem to shuttle between the nucleus and the cytoplasm. In addition, hnrnp a1 inhibits splicing of intron 9, but not intron 10. The proteins involved in the hnrnp complexes are collectively known as heterogeneous ribonucleoproteins. It has been shown that hnrnp k controls the alternative splicing of several genes, such as apoptotic peptidaseactivating factor 1 apaf1 and protein tyrosine kinase 2 ptk2b venables et al. Integrative genomewide analysis reveals cooperative. These indications that hnrnp regulated splicing is context dependent, in combination with our findings that hnrnp a2 affects cell. The alternative splicing repressors hnrnp a1a2 and ptb. As determined by the rtpcr amplification of the resulting mrna products, a timecourse coincubation of these two premrnas in a hela. Hnrnp proteins controlled by cmyc deregulate pyruvate kinase. Concentrationdependent control of pyruvate kinase m. It has been shown that hnrnp k controls the alternative splicing of several genes, such as apoptotic peptidase. The splicing factor hnrnp m is a critical regulator of.
Thus, hnrnp proteins utilize a variety of strategies to control splice site selection in a manner that is important for both alternative and constitutive premrna splicing. These results suggested that hnrnp l may control splicing from hpv16 5. The bestcharacterized hnrnps involved in splicing control are negative regulators hnrnp ab and polypyrimidine tractbinding protein ptb. Addition of the same amount of hnrnp proteins to the hnrnp ab.
After ultraviolet crosslinking, proteins were detected by. Direct interaction between hnrnpm and cdc5lplrg1 proteins. In addition to core spliceosomal proteins, splicing regulators such as sr 85 proteins and hnrnps play important roles in splice site selection and are usually targeted 86 to the splicing regulatory cis elements sre in premrna sequences wang and burge, 87 2008. Major hnrnp proteins act as general tdp43 functional. Many hnrnp proteins modulate alternative splicing of premrnas. Laboratory of biochemistry and molecular biology, rockefeller university, 1230 york avenue, new york, ny.
Concentrationdependent control of pyruvate kinase m mutually exclusive splicing by hnrnp proteins mo chen1, 2, charles j. Control of the heat stressinduced alternative splicing of. Combinatorial control of signalinduced exon repression by. Both types of hnrnp proteins have been implicated in cancer 2, 3, 18, consistent with their ability to promote expression of the protumorigenic pkm2 isoform. The antagonistic effects of general splicing factors, such as sr proteins and hnrnp proteins, can explain a large variety of splicing decisions. They include protein k and polypyrimidine tractbinding protein ptb, which is regulated by phosphorylation catalyzed by protein kinase a and is responsible for suppressing rna splicing at a particular exon by blocking access of the. Through the integration of several genomic approaches, huelga, yeo, and colleagues have identified thousands of binding sites, splicing events, and regulated genes for major hnrnp proteins a1, a2b1, f, h1, m, and u. Pdf proteins of the heterogeneous nuclear ribonucleoparticles hnrnp family form a structurally diverse group of rna binding proteins. Moreover, cooperative interactions between bound hnrnp proteins may encourage splicing between specific pairs of splice sites while simultaneously hampering other combinations. Defects in splicing lead to many human genetic diseases 24, and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer 510.
Premrna splicing is a fundamental process required for the expression of most metazoan genes 1. Evolution of sr protein and hnrnp splicing regulatory factors. These proteins are associated with premrnas in the nucleus and appear to influence premrna processing and other aspects of mrna metabolism and transport. Understanding how rna binding proteins control the splicing code is fundamental to human biology and disease. Regulatory expansion in mammals of multivalent hnrnp assemblies that globally control alternative splicing. Mechanistic control of ceacam1 splice isoforms by the heterogenous nuclear ribonuclear proteins hnrnp l, hnrnp a1 and hnrnp m kenneth j.
Structural biochemistryproteinssignaling control of. A few studies have described changes in the phosphorylation of sr or hnrnp proteins in response to cellular stress or stimulation leading to altered activity andor subcellular localization of these proteins 1, 4, 19, 20, 34. Expression of three hnrnp proteins, hnrnp a1 a1, hnrnp a2 a2 and ptb, was found to be upregulated in cancer cells by the oncogene cmyc, which promotes formation of pkm2. Pdf hnrnp proteins and splicing control researchgate. Control of the heat stressinduced alternative splicing of a. Out of all the hnrnps, a growing body of literature indicates that hnrnp a2 and itssplice variantshnrnp b1a2bb1b. Late 3splice site sa5639 is also suppressed by splicing silencers. Direct interaction between hnrnpm and cdc5l plrg1 proteins affects alternative splice site choice. Integrative genomewide analysis reveals cooperative regulation. Understanding how rna binding proteins control the splicing code. Proliferating cells and cancer cells preferentially express pkm2 over pkm1 at the protein level 1. Regulatory expansion in mammals of multivalent hnrnp. Concentrationdependent control of pyruvate kinase m mutually. Splicing phasing alternative exons original articles ying, y.
This complex of proteins bound specifically to a short rna. Whereas some studies suggested that hnrnp particles generally. Binding sites for hnrnp ab proteins stimulate the in vitro splicing of enlarged introns. Numerous sre have been defined as exonicintronic splicing enhancers. To determine if the expressed protein isozymes are a direct reflection of differences in alternative splicingas opposed to, e. Pdf 8 hnrnp proteins and splicing control benoit chabot. The function of certain sr proteins has been studied in the mouse heart through the conditional knockout approach. Using sirna screening, we found that during heat stress, alternative splicing of hsp105 was controlled by hnrnp k and psf, but not by srp38. Alternative splicing of the pyruvate kinase m pkm premrna, resulting in the exclusion of either exon 9 or 10, requires a small set of splicing factors, hnrnpa1, hnrnpa2 and ptb. Binding of hnrnp h to an exonic splicing silencer is involved. The proteins and rna regulatory elements that control tissuespecific premrna splicing in mammalian cells are mostly unknown. These rna elements consist of aurich sequences that interact with hnrnp a1hnrnp a2b1 and are located in the l1 coding region downstream of sa5639 3235. Finally, gene ontology analysis demonstrates that hnrnp a1induced splicing and gene expression targets a subset of genes with neuronal function.
To monitor splicing activity, we first used model premrnas with small introns that are spliced efficiently in vitro 7ad and 7bad. Two of these, hnrnp a1 and polypyrimidine tract binding protein ptb or hnrnp i bind to exonic splicing silencer or intronic splicing silencer elements and thus repress the splicing of certain alternatively spliced exons 10, 46. After ultraviolet crosslinking, proteins were detected by autoradiography bottom. A recent study characterizing the requirement for a range of hnrnp proteins in alternative splicing of apoptotic genes has indicated that the splicing events controlled by hnrnp proteins varies between cell lines. In vivo hnrnpmcdc5lplrg1 interactions we investigated if hnrnpm and cdc5lplrg1 proteins interact directly in vivo, by using flimfret. This control ruled out a role in general inhibition of splicing activity by these hnrnp ab proteins. Splicing is carried out by the spliceosome, which recognizes splicing signals and catalyzes the. The hnrnp proteins, as well as other nonsr splicing factors, are also subject to extensive posttranslational modifications 51. Binding of hnrnp h to an exonic splicing silencer is. However, it has been shown that hsp105 premrna is alternatively spliced in response to heat stress. Feb 23, 2012 understanding how rna binding proteins control the splicing code is fundamental to human biology and disease. Highthroughput analyses of hnrnp h1 dissects its multi. Heterogeneous nuclear ribonucleoprotein fh proteins modulate.
Many but not all major hnrnp proteins have been shown to participate in splicing control. Heterogeneous nuclear ribonucleoprotein fh proteins. Hnrnp proteins controlled by cmyc deregulate pyruvate. Hnrnp a1 controls a splicing regulatory circuit promoting mesenchymaltoepithelial transition, nucleic acids research, 20, pp. The selective degradation of many shortlived proteins in eukaryotic cells is carried out by the ubiquitin system. The hnrnp proteins have distinct nucleic acid binding properties.
Of interest, there is also the observation that the interaction between tdp43 and hnrnp proteins is highly conserved throughout evolution. However, binding of u170k to brnsd3632 representing hpv16 late 5. Here we show that intronic hnrnp abbinding sites abs can stimulate the in vitro splicing of premrnas containing artificially enlarged introns. Contextdependent control of alternative splicing by rna. C1c2 hnrnp c was identified over 30 years ago as a core component of hnrnp particles that form on all nascent transcripts4. However, although hnrnp c is one of the most abundant proteins in the nucleus, its role in splicing regulation remained unresolved. Autoregulatory feedback by rnabinding proteins oxford. Rna binding proteins that control human papillomavirus.
These altered pathways will lead to splicing differences. Relative pkm1 and pkm2 expression in tissues and cell lines correlates with hnrnp a1a2 and ptb expression. In this study, a set of proteins is identified that binds to a splicing regulatory element downstream of the neuron specific csrc nl exon. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein.
In a splicing process leading to the exclusion of exon iiic of the fgr2 gene, fox2 recruits silencing factors and assists hnrnp h1 in binding an exonic splicing silencer thereby resulting in exon exclusion. The involvement of hnrnp h in the regulation of alternative splicing may not be surprising, as hnrnp f has been shown to participate in the formation of a multiprotein complex in the dcs of csrc and play a critical role in the regulation of csrc alternative splicing min et al. The bestcharacterized hnrnps involved in splicing control are negative regulators hnrnp ab and poly pyrimidine tractbinding protein ptb. Premrna splicing is widely repressed upon heat shock in eukaryotic cells.
Multiple and specific mrna processing targets for the major. For comparison and specificity control purposes, we applied these procedures to analyze proteins associated with splice isoforms of the hnrnp ptbp1 also known as hnrnpi, with and without its mammalianspecific exon exon 9 gueroussov et al. Olson, and tom maniatisa,1 adepartment of biochemistry and molecular biophysics, columbia university college of. Heterogeneous nuclear ribonucleoparticle hnrnp proteins are among the most abundant rna binding proteins rbps and regulate cellular rna levels. Given that hnrnp f and hnrnp h each bind to the bclx b2g element with similar efficiency, cooperation between hnrnp f and hnrnp h may not be as critical for bclx splicing control. However, it is most likely that tissuespecific or developmentally regulated splicing factors have an important role in the regulation of alternative splicing, as is the case in drosophila. Using rnai screening in hela cells, we found that rna. Heterogeneous nuclear ribonucleoprotein particle ab proteins. Splicing dysregulation as oncogenic driver and passenger. In turn, the control mechanisms of alternative splicing are themselves various and complex, including the relative abundances and tissue distribution of sr and hnrnp proteins. Using rnai screening in hela cells, we found that rnabinding proteins hnrnp k and psfsfpq are necessary for the exon 12 exclusion of hsp105 during heat stress. Auagua motifs that bind proteins of the hnrnp dfamily and hnrnp a2b1 to suppress sd3632 31. Heterogeneous ribonucleoprotein particle wikipedia. Splicing activation by rbfox requires selfaggregation through its tyrosinerich domain.
As a result, the relative distribution of these regulatory proteins in the nucleus versus the cytoplasm can alter signaling pathways. The mechanisms that hnrnp a1 and ptb use to mediate splicing repression are not fully understood. Hnrnp a1 controls a splicing regulatory circuit promoting. Many rbps, among them splicing factors of the hnrnp and sr. Schematic representation of rpl3 feedback regulation mediated by hnrnp h1. Contextdependent control of alternative splicing by rnabinding. Manley1, 3 1 department of biological sciences, columbia university, new york, ny 10027 2 present addresses.
Heterogeneous nuclear ribonucleoprotein particle ab. Martinezcontreras r1, cloutier p, shkreta l, fisette jf, revil t, chabot b. Sr proteins promote the inclusion of exons during splicing, whereas hnrnp proteins repress inclusion 1. Two regulatory proteins that have their distributions regulated include srpk1 and hnrnp proteins hnrnp a1 specifically. These indications that hnrnpregulated splicing is context dependent, in combination with our findings that hnrnp a2 affects cell. These three hnrnp proteins, all previously shown to function as splicing repressors and to be overexpressed in a variety of cancers 5, 10, 18 20, exclude exon 9 by. Most studies were performed on hnrnp a1 and a2b1, which are highly expressed cellular proteins dreyfuss et al. Molecular mechanisms and implication in human pathologies chapter pdf available january 2011 with 341 reads how we measure reads. Within the downstream control element, a gggggcug motif has been shown to activate splicing of the n1 exon through its interactions with hnrnp h and f 14, 16. Gaur 1division of immunology, 2clinical and molecular pharmacology beckman research institute of the city of hope.
Alternative splicing control by polypyrimidine tract. Our future work will assess the individual or cooperative nature of hnrnp f and hmediated control of bclx splicing. Sr proteins and hnrnps can compete for binding to cisregulatory rna. The mechanism by which this is accomplished is unclear, as highaffinity hnrnp abbinding sites do not often overlap. Rna binding proteins that control human papillomavirus gene. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle hnrnp proteins, among the most abundant rna binding proteins, coordinate to regulate alternative premrna splicing as in human cells.