Examining SRP pathway function in mRNA localization to the endoplasmic reticulum.

Signal recognition particle (SRP) pathway function in protein translocation across the endoplasmic reticulum (ER) is well established; its role in RNA localization to the ER remains, however, unclear. In current models, mRNAs undergo translation- and SRP-dependent trafficking to the ER, with ER localization mediated via interactions between SRP-bound translating ribosomes and the ER-resident SRP receptor (SR), a heterodimeric complex comprising SRA, the SRP-binding subunit, and SRB, an integral membrane ER protein. To study SRP pathway function in RNA localization, SR knockout (KO) mammalian cell lines were generated and the consequences of SR KO on steady-state and dynamic mRNA localization examined. CRISPR/Cas9-mediated SRPRB KO resulted in profound destabilization of SRA. Pairing siRNA silencing of SRPRA in SRPRB KO cells yielded viable SR KO cells. Steady-state mRNA compositions and ER-localization patterns in parental and SR KO cells were determined by cell fractionation and deep sequencing. Notably, steady-state cytosol and ER mRNA compositions and partitioning patterns were largely unaltered by loss of SR expression. To examine SRP pathway function in RNA localization dynamics, the subcellular trafficking itineraries of newly exported mRNAs were determined by 4-thiouridine (4SU) pulse-labeling/4SU-seq/cell fractionation. Newly exported mRNAs were distinguished by high ER enrichment, with ER localization being SR-independent. Intriguingly, under conditions of translation initiation inhibition, the ER was the default localization site for all newly exported mRNAs. These data demonstrate that mRNA localization to the ER can be uncoupled from the SRP pathway function and reopen questions regarding the mechanism of RNA localization to the ER.

Quantitative Proteomics Links the LRRC59 Interactome to mRNA Translation on the ER Membrane.

Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61ß (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61ß and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [35S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation.

The transcriptional repressor BCL11A promotes breast cancer metastasis.

The phenotypes of each breast cancer subtype are defined by their transcriptomes. However, the transcription factors that regulate differential patterns of gene expression that contribute to specific disease outcomes are not well understood. Here, using gene silencing and overexpression approaches, RNA-Seq, and splicing analysis, we report that the transcription factor B-cell leukemia/lymphoma 11A (BCL11A) is highly expressed in triple-negative breast cancer (TNBC) and drives metastatic disease. Moreover, BCL11A promotes cancer cell invasion by suppressing the expression of muscleblind-like splicing regulator 1 (MBNL1), a splicing regulator that suppresses metastasis. This ultimately increases the levels of an alternatively spliced isoform of integrin-α6 (ITGA6), which is associated with worse patient outcomes. These results suggest that BCL11A sustains TNBC cell invasion and metastatic growth by repressing MBNL1-directed splicing of ITGA6 Our findings also indicate that BCL11A lies at the interface of transcription and splicing and promotes aggressive TNBC phenotypes.

Mapping transcriptome-wide protein-RNA interactions to elucidate RNA regulatory programs.

BACKGROUND: Our understanding of post-transcriptional gene regulation has increased exponentially with the development of robust methods to define protein-RNA interactions across the transcriptome. In this review, we highlight the evolution and successful applications of crosslinking and immunoprecipitation (CLIP) methods to interrogate protein-RNA interactions in a transcriptome-wide manner. RESULTS: Here, we survey the vast array of in vitro and in vivo approaches used to identify protein-RNA interactions, including but not limited to electrophoretic mobility shift assays, systematic evolution of ligands by exponential enrichment (SELEX), and RIP-seq. We particularly emphasize the advancement of CLIP technologies, and detail protocol improvements and computational tools used to analyze the output data. Importantly, we discuss how profiling protein-RNA interactions can delineate biological functions including splicing regulation, alternative polyadenylation, cytoplasmic decay substrates, and miRNA targets. CONCLUSIONS: In summary, this review summarizes the benefits of characterizing RNA-protein networks to further understand the regulation of gene expression and disease pathogenesis. Our review comments on how future CLIP technologies can be adapted to address outstanding questions related to many aspects of RNA metabolism and further advance our understanding of RNA biology.

DAZL Regulates Germ Cell Survival through a Network of PolyA-Proximal mRNA Interactions.

The RNA binding protein DAZL is essential for gametogenesis, but its direct in vivo functions, RNA targets, and the molecular basis for germ cell loss in Dazl-null mice are unknown. Here, we mapped transcriptome-wide DAZL-RNA interactions in vivo, revealing DAZL binding to thousands of mRNAs via polyA-proximal 3' UTR interactions. In parallel, fluorescence-activated cell sorting and RNA-seq identified mRNAs sensitive to DAZL deletion in male germ cells. Despite binding a broad set of mRNAs, integrative analyses indicate that DAZL post-transcriptionally controls only a subset of its mRNA targets, namely those corresponding to a network of genes that are critical for germ cell proliferation and survival. In addition, we provide evidence that polyA sequences have key roles in specifying DAZL-RNA interactions across the transcriptome. Our results reveal a mechanism for DAZL-RNA binding and illustrate that DAZL functions as a master regulator of a post-transcriptional mRNA program essential for germ cell survival.

Ptbp2 Controls an Alternative Splicing Network Required for Cell Communication during Spermatogenesis.

Alternative splicing has essential roles in development. Remarkably, spermatogenic cells express more alternatively spliced RNAs compared to most whole tissues; however, regulation of these RNAs remains unclear. Here, we characterize the alternative splicing landscape during spermatogenesis and reveal an essential function for the RNA-binding protein Ptbp2 in this highly regulated developmental program. We found that Ptbp2 controls a network of genes involved in cell adhesion, migration, and polarity, suggesting that splicing regulation by Ptbp2 is critical for germ cell communication with Sertoli cells (multifunctional somatic cells necessary for spermatogenesis). Indeed, Ptbp2 ablation in germ cells resulted in disorganization of the filamentous actin (F-actin) cytoskeleton in Sertoli cells, indicating that alternative splicing regulation is necessary for cellular crosstalk during germ cell development. Collectively, the data delineate an alternative splicing regulatory network essential for spermatogenesis, the splicing factor that controls it, and its biological importance in germ-Sertoli communication.

RNA Binding Protein Ptbp2 Is Essential for Male Germ Cell Development.

RNA binding proteins (RBPs) are increasingly recognized as essential factors in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. In the nervous system, the function and importance of PTB protein 2 (Ptbp2) as a key alternative splicing regulator is well established. Ptbp2 is also abundantly expressed during spermatogenesis, but its role in this developmental program has not been explored. Additionally, the importance of alternative splicing regulation in spermatogenesis is unclear. Here, we demonstrate that Ptbp2 is essential for spermatogenesis. We also describe an improved dual fluorescence flow cytometry strategy to discriminate, quantify, and collect germ cells in different stages of development. Using this approach, in combination with traditional histological methods, we show that Ptbp2 ablation results in germ cell loss due to increased apoptosis of meiotic spermatocytes and postmeiotic arrest of spermatid differentiation. Furthermore, we show that Ptbp2 is required for alternative splicing regulation in the testis, as in brain. Strikingly, not all of the alternatively spliced RNAs examined were sensitive to Ptbp2 loss in both tissues. Collectively, the data provide evidence for an important role for alternative splicing regulation in germ cell development and a central role for Ptbp2 in this process.