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Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While “exitrons” are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed “falsitrons,” that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.
Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends.
Lieblingsbild
(2017)
Dieses Bild ist wichtig, weil wir daran verstanden haben, wie in der Zelle fehlerhaftes Spleißen verhindert wird. Dazu muss man wissen, dass unsere Gene sich aus Exons und dazwischenliegenden Introns zusammensetzen. Während des Spleißens werden die Introns entfernt und die Exons in ein reifes Transkript zusammengefügt, das dann für ein Protein kodiert. Allerdings gibt es innerhalb der Introns viele Bereiche, die einem Exon sehr ähnlich sehen. Werden diese sogenannten "PseudoExons" fälschlicherweise während des Spleißprozesses erkannt und in das reife Transkript eingebaut, kann das fatale Folgen für das kodierte Protein und oft die gesamte Zelle haben. ...
SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.
Macrophages exert the primary cellular immune response. Pathogen components like bacterial lipopolysaccharides (LPS) stimulate macrophage migration, phagocytotic activity and cytokine expression. Previously, we identified the poly(A)+ RNA interactome of RAW 264.7 macrophages. Of the 402 RNA-binding proteins (RBPs), 32 were classified as unique in macrophages, including nineteen not reported to interact with nucleic acids before. Remarkably, P23 a HSP90 co-chaperone, also known as cytosolic prostaglandin E2 synthase (PTGES3), exhibited differential poly(A)+ RNA binding in untreated and LPS-induced macrophages. To identify mRNAs bound by P23 and to elucidate potential regulatory RBP functions in macrophages, we immunoprecipitated P23 from cytoplasmic extracts of cross-linked untreated and LPS-induced cells. RNAseq revealed that enrichment of 44 mRNAs was reduced in response to LPS. Kif15 mRNA, which encodes kinesin family member 15 (KIF15), a motor protein implicated in cytoskeletal reorganization and cell mobility was selected for further analysis. Noteworthy, phagocytic activity of LPS-induced macrophages was enhanced by P23 depletion. Specifically, in untreated RAW 264.7 macrophages, decreased P23 results in Kif15 mRNA destabilization, diminished KIF15 expression and accelerated macrophage migration. We show that the unexpected RBP function of P23 contributes to the regulation of macrophage phagocytotic activity and migration.
The 3′ untranslated regions (3′ UTRs) of transcripts serve as important hubs for posttranscriptional gene expression regulation. Here, we find that the exonisation of intergenic Alu elements introduced new terminal exons and polyadenylation sites during human genome evolution. While Alu exonisation from introns has been described previously, we shed light on a novel mechanism to create alternative 3′ UTRs, thereby opening opportunities for differential posttranscriptional regulation. On the mechanistic level, we show that intergenic Alu exonisation can compete both with alternative splicing and polyadenylation in the upstream gene. Notably, the Alu-derived isoforms are often expressed in a tissue-specific manner, and the Alu-derived 3′ UTRs can alter mRNA stability. In summary, we demonstrate that intergenic elements can affect processing of preceding genes, and elucidate how intergenic Alu exonisation can contribute to tissue-specific posttranscriptional regulation by expanding the repertoire of 3′ UTRs.
Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.
Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3’ UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells we show that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. We observe increased association of the translational repressor Bru1 with osk mRNA upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3’ UTR.
Precise knowledge on the binding sites of an RNA-binding protein (RBP) is key to understanding the complex post-transcriptional regulation of gene expression. This information can be obtained from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments. Here, we present a complete data analysis workflow to reliably detect RBP binding sites from iCLIP data. The workflow covers all steps from the initial quality control of the sequencing reads up to peak calling and quantification of RBP binding. For each tool, we explain the specific requirements for iCLIP data analysis and suggest optimised parameter settings.
mRNA localization to subcellular compartments has been reported across all kingdoms of life and it is generally believed to promote asymmetric protein synthesis and localization. In striking contrast to previous observations, we show that in S. cerevisiae the B-type cyclin CLB2 mRNA is localized and translated in the yeast bud, while the Clb2 protein, a key regulator of mitosis progression, is concentrated in the mother nucleus. Using single-molecule RNA imaging in fixed (smFISH) and living cells (MS2 system), we show that the CLB2 mRNA is transported to the yeast bud by the She2-She3 complex, via an mRNA ZIP-code situated in the coding sequence. In CLB2 mRNA localization mutants, Clb2 protein synthesis in the bud is decreased resulting in changes in cell cycle distribution and genetic instability. Altogether, we propose that CLB2 mRNA localization acts as a sensor for bud development to couple cell growth and cell cycle progression, revealing a novel function for mRNA localization.