Position-dependent activity of CELF2 in the regulation of splicing and implications for signal-responsive regulation in T cells.

CELF2 is an RNA binding protein that has been implicated in developmental and signal-dependent splicing in the heart, brain and T cells. In the heart, CELF2 expression decreases during development, while in T cells CELF2 expression increases both during development and in response to antigen-induced signaling events. Although hundreds of CELF2-responsive splicing events have been identified in both heart and T cells, the way in which CELF2 functions has not been broadly investigated. Here we use CLIP-Seq to identified physical targets of CELF2 in a cultured human T cell line. By comparing the results with known functional targets of CELF2 splicing regulation from the same cell line we demonstrate a generalizable position-dependence of CELF2 activity that is consistent with previous mechanistic studies of individual CELF2 target genes in heart and brain. Strikingly, this general position-dependence is sufficient to explain the bi-directional activity of CELF2 on 2 T cell targets recently reported. Therefore, we propose that the location of CELF2 binding around an exon is a primary predictor of CELF2 function in a broad range of cellular contexts.

Stem-loop recognition by DDX17 facilitates miRNA processing and antiviral defense.

DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements.

Transcriptome-wide RNA interaction profiling reveals physical and functional targets of hnRNP L in human T cells.

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 high-throughput 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.

Polyadenylation site-specific differences in the activity of the neuronal ßCstF-64 protein in PC-12 cells.

Recent genome-wide analyses have implicated alternative polyadenylation - the process of regulated mRNA 3' end formation - as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, ßCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that ßCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for ßCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that ßCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that ßCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the ß-adducin mRNA. Notably, we demonstrate that the activity of ßCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the ßCstF-64 protein. Our data address the polyadenylation functions of ßCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.

Minimal functional domains of paralogues hnRNP L and hnRNP LL exhibit mechanistic differences in exonic splicing repression.

Understanding functional distinctions between related splicing regulatory proteins is critical to deciphering tissue-specific control of alternative splicing. The hnRNP (heterogeneous nuclear ribonucleoprotein) L and hnRNP LL (hnRNP L-like) proteins are paralogues that have overlapping, but distinct, expression patterns and functional consequences. These two proteins share high sequence similarity in their RRMs (RNA-recognition motifs), but diverge in regions outside of the RRMs. In the present study, we use an MS2-tethering assay to delineate the minimal domains of hnRNP L and hnRNP LL which are required for repressing exon inclusion. We demonstrate that for both proteins, regions outside the RRMs, the N-terminal region, and a linker sequence between RRMs 2 and 3, are necessary for exon repression, but are only sufficient for repression in the case of hnRNP LL. In addition, both proteins require at least one RRM for maximal repression. Notably, we demonstrate that the region encompassing RRMs 1 and 2 of hnRNP LL imparts a second silencing activity not observed for hnRNP L. This additional functional component of hnRNP LL is consistent with the fact that the full-length hnRNP LL has a greater silencing activity than hnRNP L. Thus the results of the present study provide important insight into the functional and mechanistic variations that can exist between two highly related hnRNP proteins.

hnRNP L and hnRNP A1 induce extended U1 snRNA interactions with an exon to repress spliceosome assembly.

Pre-mRNA splicing is catalyzed through the activity of the spliceosome, a dynamic enzymatic complex. Forcing aberrant interactions within the spliceosome can reduce splicing efficiency and alter splice site choice; however, it is unknown whether such alterations are naturally exploited mechanisms of splicing regulation. Here, we demonstrate that hnRNP L represses CD45 exon 4 by recruiting hnRNP A1 to a sequence upstream of the 5' splice site. Together, hnRNP L and A1 induce extended contacts between the 5' splice site-bound U1 snRNA and neighboring exonic sequences that, in turn, inhibit stable association of U6 snRNA and subsequent catalysis. Importantly, analysis of several exons regulated by hnRNP L shows a clear relationship between the potential for binding of hnRNP A1 and U1 snRNA and the effect of hnRNP L on splicing. Together, our results demonstrate that conformational perturbations within the spliceosome are a naturally occurring and generalizable mechanism for controlling alternative splicing decisions.

The τCstF-64 polyadenylation protein controls genome expression in testis.

The τCstF-64 polyadenylation protein (gene symbol Cstf2t) is a testis-expressed orthologue of CstF-64. Mice in which Cstf2t was knocked out had a phenotype that was only detected in meiotic and postmeiotic male germ cells, giving us the opportunity to examine CstF-64 function in an isolated developmental system. We performed massively parallel clonally amplified sequencing of cDNAs from testes of wild type and Cstf2t(-/-) mice. These results revealed that loss of τCstF-64 resulted in large-scale changes in patterns of genome expression. We determined that there was a significant overrepresentation of RNAs from introns and intergenic regions in testes of Cstf2t(-/-) mice, and a concomitant use of more distal polyadenylation sites. We observed this effect particularly in intronless small genes, many of which are expressed retroposons that likely co-evolved with τCstF-64. Finally, we observed overexpression of long interspersed nuclear element (LINE) sequences in Cstf2t(-/-) testes. These results suggest that τCstF-64 plays a role in 3' end determination and transcription termination for a large range of germ cell-expressed genes.

Living or dying by RNA processing: caspase expression in NSCLC.

Protein expression in humans is controlled by numerous RNA processing steps that occur between transcription of a gene and translation of protein. However, the importance of such regulatory steps to human diseases, especially cancer, is just now coming to light. Changes in the alternative splicing or stability of mRNA transcribed from genes involved in cell-cycle control, cell proliferation, and apoptosis has been linked to tumor formation and progression. Nevertheless, in the majority of these cases, the identity of the regulators that control the expression of such cancer-related genes is poorly understood. In this issue of the JCI, Goehe et al. demonstrate that heterogeneous nuclear ribonuclear protein family member L (hnRNP L), a member of the hnRNP family of RNA processing factors, is specifically phosphorylated in non-small cell lung cancer (NSCLC). The phosphorylated hnRNP L, in turn, promotes expression of the antiapoptotic form of caspase-9, thereby contributing to tumorigenesis.

A family of splice variants of CstF-64 expressed in vertebrate nervous systems.

BACKGROUND: Alternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs. RESULTS: We discovered several closely related splice variants of CstF-64 - collectively called betaCstF-64 - that could potentially contribute to proteomic diversity in the nervous system. The betaCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major betaCstF-64 variant mRNA is generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of betaCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of betaCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to betaCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since betaCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that betaCstF-64 has an evolutionarily conserved function in these animals. betaCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for betaCstF-64 in neural gene expression throughout development. Finally, experiments in representative cell lines suggest that betaCstF-64 is expressed in neurons but not glia. CONCLUSION: This is the first report of a family of splice variants encoding a key polyadenylation protein that is expressed in a nervous system-specific manner. We propose that betaCstF-64 contributes to proteomic diversity by regulating alternative polyadenylation of neural mRNAs.