A Conserved Serine of Heterogeneous Nuclear Ribonucleoprotein L (hnRNP L) Mediates Depolarization-regulated Alternative Splicing of Potassium Channels

Liu, Guodong; Razanau, Aleh; Yan, Hai; Yu, Jiankun; Sohail, Muhammad; Lobo, Vincent G.; Chu, Junfeng; Kung, Sam K. P.; Xie, Jiuyong

doi:10.1074/jbc.m112.357343

Cited by 69 publications

(78 citation statements)

References 37 publications

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“…We also note the possibility that stimulation of T cells results in a posttranslational modification(s) of hnRNP L that alters its binding affinity and/or specificity. While such regulation of hnRNP L binding has not been reported in T cells, at least two reports have suggested that phosphorylation of hnRNP L in other cell types can alter its ability to recognize specific RNA target sequences (56,57). We emphasize, however, that less than 10% of the total binding events we detect for hnRNP L appear to be condition specific, and this number decreases further with increased stringency of peak calling (see Table S7 in the supplemental material).…”

Section: Discussionmentioning

confidence: 50%

Transcriptome-Wide RNA Interaction Profiling Reveals Physical and Functional Targets of hnRNP L in Human T Cells

Shankarling

Cole

Mallory

et al. 2014

Molecular and Cellular Biology

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The RNA processing factor hnRNP L is required for T cell development and function. However, the spectrum of direct targets of hnRNP L activity in T cells has yet to be defined. In this study, we used cross-linking and immunoprecipitation followed by highthroughput sequencing (CLIP-seq) to identify the RNA binding sites of hnRNP L within the transcriptomes of human CD4؉ and cultured Jurkat T cells. We find that hnRNP L binds preferentially to transcripts encoding proteins involved in RNA processing and in Wnt and T cell receptor (TCR) signaling. This binding is largely conserved across both quiescent and activated T cells, in agreement with the critical role of hnRNP L throughout T cell biology. Importantly, based on the binding profile of hnRNP L, we validate numerous instances of hnRNP L-dependent alternative splicing of genes critical to T cell function. We further show that alternative exons with weak 5= splice site sequences specifically show a strong correlation between hnRNP L binding and hnRNP L-dependent splicing regulation. Together, these data provide the first transcriptome-wide analysis of the RNA targets of hnRNP L in lymphoid cells and add to the functional understanding of hnRNP L in human biology. RNA-based gene regulation encompasses many universal processes that are essential to shaping the composition and function of the proteome in eukaryotic cells (1). In particular, mechanisms such as alternative splicing, alternative 3=-end processing, and microRNA (miRNA)-directed processes control not only the level of expression of a transcript but also the distinct protein isoforms encoded by a given gene. Therefore, such regulatory mechanisms allow for both the expansion and the control of genetic information.Virtually all processes of RNA-based gene regulation are controlled by the activity of a family of RNA binding proteins known as hnRNPs (heterogeneous nuclear ribonucleoproteins) (2-5). Most members of the hnRNP family are ubiquitously expressed and bind to RNA substrates through RRM (RNA recognition motif) or KH (hnRNP K homology) domains (4). Depending on the location of binding and associated proteins, hnRNPs have been shown to either enhance or repress the inclusion of particular exons, promote or inhibit splicing efficiency, alter the use of competing 3= cleavage and polyadenylation sites, control mRNA stability, and regulate miRNA access to target genes (2-5). All hnRNPs that have been well studied appear to be capable of carrying out all of these activities; therefore, the location of binding appears to be a primary determinant of whether and how a specific hnRNP controls the expression of a particular gene (2-4, 6).Given the intricacy of T cell development and function, it is not surprising that RNA-based gene regulation is increasingly recognized as a critical determinant of the growth and activity of T cells (7,8). In particular, one hnRNP for which there is much evidence of a functional role in T cell biology is hnRNP L (9-12). hnRNP L is a 65-kDa hnRNP family member that contains 4 RR...

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Section: Discussionmentioning

confidence: 50%

Transcriptome-Wide RNA Interaction Profiling Reveals Physical and Functional Targets of hnRNP L in Human T Cells

Shankarling

Cole

Mallory

et al. 2014

Molecular and Cellular Biology

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“…Membrane depolarization of cerebellar neurons induces phosphorylation of hnRNP L at Ser513 by CaMKIV, which enhances the interaction of hnRNP L with the CaMKIVresponsive RNA element (CaRRE1) upstream of STREX. As a result, U2AF binding is reduced and STREX is skipped (44). Second, histone hyperacetylation can cause up-regulation of specific splicing regulators at the transcription level, which will lead to either inclusion or skipping of their target alternative exons (Fig.…”

Section: Discussionmentioning

confidence: 99%

Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes

Sharma¹,

Nguyen²,

Geng³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Here we report that, in mouse primary or embryonic stem cell-derived cardiomyocytes, increased calcium levels induce robust and reversible skipping of several alternative exons from endogenously expressed genes. Interestingly, we demonstrate a calcium-mediated splicing regulatory mechanism that depends on changes of histone modifications. Specifically, the regulation occurs through changes in calciumresponsive kinase activities that lead to alterations in histone modifications and subsequent changes in the transcriptional elongation rate and exon skipping. We demonstrate that increased intracellular calcium levels lead to histone hyperacetylation along the body of the genes containing calcium-responsive alternative exons by disrupting the histone deacetylase-to-histone acetyltransferase balance in the nucleus. Consequently, the RNA polymerase II elongation rate increases significantly on those genes, resulting in skipping of the alternative exons. These studies reveal a mechanism by which calcium-level changes in cardiomyocytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns.alternative splicing | histone hyperacetylation | transcriptional elongation rate | calcium | cardiomyocytes A lternative splicing is a robust mechanism that regulates the functional output of a genome. More than 90% of human protein-coding genes undergo alternative splicing, which significantly increases proteomic complexity of the human genome (1, 2). The precise spatial-temporal regulation of alternative splicing plays a crucial role in controlling gene expression. Decades of studies have yielded a wealth of knowledge on how tissue-and developmental stage-specific alternative splicing is regulated (3). However, signaling-controlled alternative splicing in response to environmental cues has attracted far less attention, and regulatory mechanistic insights have only begun to emerge in recent years (4).Calcium is an important intracellular second messenger that regulates many biological processes. Although most studies have focused on how calcium regulates gene expression at the transcriptional level (5, 6), a small number of studies have also explored how changes in intracellular calcium levels can lead to alternative splicing pattern alterations (7-12). For example, in neuronal cells, a splicing-sensitive exon array identified more than 5,000 genes that change their splicing pattern in response to elevated calcium levels (13). However, because only a handful of studies have investigated the underlying mechanisms, it remains largely unknown how calcium-induced alternative splicing changes are regulated. These studies have revealed two distinct mechanisms by which calcium-responsive alternative exons can be regulated: one RNA-binding protein (RBP)-dependent and the other RBP-independent (14, 15).Several studies using depolarized...

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“…However, we have found a CA-rich splicing regulatory element called CaRRE1 at this location (12)(13)(14)(15), suggesting relaxation of the constraint in some transcripts and the potential existence of other, similar elements. In particular, a purine-rich (Grich or A-rich) element such as a G tract at this location is expected to strongly disrupt the 3= splice site (3=SS).…”

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confidence: 80%

“…Immunodepletion of hnRNP H/F from a HeLa nuclear extract containing 0.5 M NaCl was carried out based on a published procedure (51). Immunodepleted HeLa nuclear extract was dialyzed 3 times against DG buffer (12) Purification of recombinant His-hnRNP H. To purify the recombinant N-terminal histidine-tagged hnRNP H, Escherichia coli Rosetta-gami 2 (DE3) pLysS (Novagen) bacteria transformed with pET28a-hnRNP H were grown overnight in LB medium containing 50 g/ml kanamycin and induced with 0.3 mM isopropyl 1-thio-␤-D-galactopyranoside at 37°C for 3 h. Protein purification was carried out as described previously (17) except that phosphate-buffered saline (PBS) was used instead of 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

Evolutionary Emergence of a Novel Splice Variant with an Opposite Effect on the Cell Cycle

Sohail

Xie

2015

Molecular and Cellular Biology

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Alternative splicing contributes greatly to the diversification of mammalian proteomes, but the molecular basis for the evolutionary emergence of splice variants remains poorly understood. We have recently found a novel class of splicing regulatory elements between the polypyrimidine tract (Py) and 3= AG (REPA) at intron ends in many human genes, including the multifunctional PRMT5 (for protein arginine methyltransferase 5) gene. The PRMT5 element is comprised of two G tracts that arise in most mammals and accompany significant exon skipping in human transcripts. The G tracts inhibit splicing by recruiting heterogeneous nuclear ribonucleoprotein (hnRNP) H and F (H/F) to reduce U2AF65 binding to the Py, causing exon skipping. The resulting novel shorter variant PRMT5S exhibits a histone H4R3 methylation effect similar to that seen with the original longer PRMT5L isoform but exhibits a distinct localization and preferential control of critical genes for cell cycle arrest at interphase in comparison to PRMT5L. This report thus provides a molecular mechanism for the evolutionary emergence of a novel splice variant with an opposite function in a fundamental cell process. The presence of REPA elements in a large group of genes implies their wider impact on different cellular processes for increased protein diversity in humans.A lternative precursor mRNA (pre-mRNA) splicing greatly increases the proteomic diversity in metazoans (1-3). In particular, splice variants have reached the highest complexity in humans and other primates (4, 5), with about 90% of human genes alternatively spliced (6, 7). Aberrant splicing causes a large fraction of human genetic diseases (8, 9). However, the molecular basis for the evolutionary emergence of alternative exons that impact protein functions and cellular processes remains largely unknown, although several models have been proposed (10).The 3= end of introns between the polypyrimidine tract (Py) and 3=AG is highly constrained in sequence and length, with a consensus sequence of PyNYAG (Y, pyrimidine; N, any nucleotide) (11). However, we have found a CA-rich splicing regulatory element called CaRRE1 at this location (12-15), suggesting relaxation of the constraint in some transcripts and the potential existence of other, similar elements. In particular, a purine-rich (Grich or A-rich) element such as a G tract at this location is expected to strongly disrupt the 3= splice site (3=SS).G tracts with a minimal functional GGG motif are splicing regulatory elements bound by heterogeneous nuclear ribonucleoprotein (hnRNP) H (H1) or its paralogues, including hnRNP F (16-24). They are enhancers or silencers of splicing, depending on their location in the pre-mRNA (17, 22, 24-28). We have identified G tracts between the Py and 3= AG in more than a thousand human genes, including PRMT5 (for protein arginine methyltransferase 5). We call elements at this location REPA (regulatory elements between the Py and 3=AG) (29). These REPA G tracts appear to have mostly emerged in mammalian ancestors to ...

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A Conserved Serine of Heterogeneous Nuclear Ribonucleoprotein L (hnRNP L) Mediates Depolarization-regulated Alternative Splicing of Potassium Channels

Cited by 69 publications

References 37 publications

Transcriptome-Wide RNA Interaction Profiling Reveals Physical and Functional Targets of hnRNP L in Human T Cells

Transcriptome-Wide RNA Interaction Profiling Reveals Physical and Functional Targets of hnRNP L in Human T Cells

Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes

Evolutionary Emergence of a Novel Splice Variant with an Opposite Effect on the Cell Cycle

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