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

Abstract

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...

RNA-binding protein hnRNPLL regulates mRNA splicing and stability during B-cell to plasma-cell differentiation

“…hnRNPLL is structurally similar to hnRNPL; moreover, the (CA)n RNA motif we have identified here as recognized by hnRNPLL is similar to the motif identified through SELEX experiments as recognized by hnRNPL (6,21,35). Thus, these two related RBPs may have redundant as well as nonoverlapping functions, as confirmed by the overlap in their target genes (e.g., NFAT5) (this work and ref.…”

“…As observed for many other RBPs (1,2,6,14), the motifs recognized by hnRNPLL are rather degenerate in their sequence. An interesting question is how, given this degeneracy, hnRNPLL achieves specific recognition of its target pre-mRNAs.…”

“…Thus, these two related RBPs may have redundant as well as nonoverlapping functions, as confirmed by the overlap in their target genes (e.g., NFAT5) (this work and ref. 6). It will be interesting to examine further the shared vs. independent functions of hnRNPLL and hnRNP L in lymphocytes and other cell types (1).…”

“…Plasma-cell differentiation is also accompanied by alteration of mRNA alternative processing: The mRNA encoding the transmembrane phosphatase CD45 undergoes alternative splicing to exclude exons 4-6, thus switching the CD45 protein from its highest-molecular-weight isoform, CD45RABC (also known as B220 in B cells), to the lowest-molecular-weight isoform, CD45RO (17,18). However, the role of posttranscriptional regulation in plasma-cell differentiation is less well characterized than the analogous process in T cells (1,6,(9)(10)(11)19).…”

“…RNAprotein interactions have been explored through RNA crosslinking and immunoprecipitation (RNA-CLIP), a method that uses UV irradiation to cross-link RNA to its binding proteins, followed by immunoprecipitation of selected RBPs and nextgeneration sequencing to identify RNAs bound to those RBPs at a genome-/transcriptome-wide level (3)(4)(5)(6)(7). A recent improvement known as PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation) uses a photoactive ribonucleotide analog to increase the efficiency of RNA/protein crosslinking (8).…”