Heterogeneous Nuclear Ribonucleoprotein G Regulates Splice Site Selection by Binding to CC(A/C)-rich Regions in Pre-mRNA

Abstract

Almost every protein-coding gene undergoes pre-mRNA splicing, and the majority of these pre-mRNAs are alternatively spliced. Alternative exon usage is regulated by the transient formation of protein complexes on the pre-mRNA that typically contain heterogeneous nuclear ribonucleoproteins (hnRNPs). Here we characterize hnRNP G, a member of the hnRNP class of proteins. We show that hnRNP G is a nuclear protein that is expressed in different concentrations in various tissues and that interacts with other splicing regulatory proteins. hnRNP G is part of the supraspliceosome, where it regulates alternative splice site selection in a concentrationdependent manner. Its action on alternative exons can occur without a functional RNA-recognition motif by binding to other splicing regulatory proteins. The RNA-recognition motif of hnRNP G binds to a loose consensus sequence containing a CC(A/C) motif, and hnRNP G preferentially regulates alternative exons where this motif is clustered in close proximity. The X-chromosomally encoded hnRNP G regulates different RNAs than its Y-chromosomal paralogue RNA-binding motif protein, Y-linked (RBMY), suggesting that differences in alternative splicing, evoked by the sexspecific expression of hnRNP G and RBMY, could contribute to molecular sex differences in mammals.All protein-coding genes undergo pre-mRNA processing, and the large majority of these genes are alternatively spliced (1). Alternative exons can change many functional aspects of mRNAs and their encoded proteins. The best understood functions are stop codons or frameshifts that are introduced by 20 -35% of alternative exons, which often destine the altered mRNA to nonsense-mediated decay. Examples described in the literature show that alternative splicing regulates the binding properties, intracellular localization, enzymatic activity, protein stability, and post-translational modifications of a large number of proteins (reviewed in Ref.2). Thus, it appears that alternative pre-mRNA processing is a key mechanism regulating the gene expression of complex organisms by generating multiple mRNA isoforms, which encode functionally diverse proteins. Despite its importance, the exact mechanisms governing splice site selection are still poorly understood. In vertebrate systems, protein complexes assemble transiently on exons, and their interaction with the splicing machinery as well as RNA-RNA interactions between spliceosomal proteins and pre-mRNA determine whether an exon is included or skipped (reviewed in Refs. 3 and 4).When isolated from nuclei of mammalian cells, RNA polymerase II transcripts are found assembled in large ribonucleoprotein 21-MDa complexes, the supraspliceosome, composed of all five spliceosomal small nuclear ribonucleoproteins as well as additional proteins. The entire repertoire of nuclear pre-mRNAs, independent of their length or number of introns, is individually found assembled in supraspliceosomes (reviewed in Ref. 5). Structural studies revealed that the supraspliceosome is composed of four substructure...

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

“…In previous experiments, fractions corresponding to the analogous positions in the gradient were enriched for the five spliceosomal U snRNAs (37,(39)(40)(41) (Fig. S1D), various pre-mRNAs (36,42,43), and regulatory splicing factors (41,44,45) (Fig. 1E) and contained active spliceosomes (37).…”

Expression levels of hnRNP G and hTra2‐beta1 correlate with opposite outcomes in endometrial cancer biology

“…As it is well known that many alternative splicing factors, including hnRNP G and hTra2-beta1, are shuttling intracellularly between nucleus and cytoplasm, it is of large interest to further investigate the mechanism which influences or actively regulates cellular splicing factors shuttling. Both hnRNP G and hTra2-beta1 are sequence-specific splicing factors 21,25,37 and are potentially capable to exert opposite effects on expression of alternative spliced genes in a concentration-dependent manner. 25 HnRNP G preferentially regulates alternative exons where a AAGU or CC(A/C) containing motif is clustered.…”

“…25 HnRNP G preferentially regulates alternative exons where a AAGU or CC(A/C) containing motif is clustered. 25,37 A panel of tumor biological important genes are regulated through hTra2-beta1 mediated GAA-rich enhancer-dependent splicing. 21 CD44, ESRa, BRCA1, scr, Her2/neu are such genes regulated by hTra2-beta1, which play important roles especially in hormone-dependent malignancies such as breast and ovarian cancer.…”

X-linked RNA-binding motif protein (RBMX) is required for the maintenance of Borna disease virus nuclear viral factories

“…In this study, we focused on the host factors that associate with host chromosomes, because our previous study demonstrated that the formation of vSPOTs, as well as the dynamics of viral RNPs, appeared to be associated with the structural dynamics of chromosomes, such as mitosis (Heinrich et al, 2009;Matsumoto et al, 2012). Among the candidate host factors (Table S1, available in the online Supplementary Material), we focused on a nuclear protein, RBMX, because only this protein has been shown to be associated with chromosomal structure and to form nuclear foci (Heinrich et al, 2009).…”

“…Among the candidate host factors (Table S1, available in the online Supplementary Material), we focused on a nuclear protein, RBMX, because only this protein has been shown to be associated with chromosomal structure and to form nuclear foci (Heinrich et al, 2009). RBMX is an RNA-binding protein that participates in the spliceosome and plays a role in alternative splicing (Heinrich et al, 2009).…”