Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1

Alternative splicing is controlled by differential binding of trans-acting RNA binding proteins (RBPs) to cis-regulatory elements in intronic and exonic pre-mRNA regions. How secondary structure in the pre-mRNA transcripts affects recognition by RBPs and determines alternative exon usage is poorly understood. The MALT1 paracaspase is a key component of signaling pathways that mediate innate and adaptive immune responses. Alternative splicing of MALT1 exon7 is critical for controlling optimal T cell activation. Here, we demonstrate that processing of the MALT1 pre-mRNA depends on RNA structural elements that shield the 5′ and 3′ splice sites of the alternatively spliced exon7. By combining biochemical analyses with chemical probing and NMR we show that the RBPs hnRNP U and hnRNP L bind competitively and with comparable affinities to identical stem-loop RNA structures flanking the 5′ and 3′ splice sites of MALT1 exon7. While hnRNP U stabilizes RNA stem-loop conformations that maintain exon7 skipping, hnRNP L unwinds these RNA elements to facilitate recruitment of the essential splicing factor U2AF2 to promote exon7 inclusion. Our data represent a paradigm for the control of splice site selection by differential RBP binding and modulation of pre-mRNA structure.

hnRNP U is differentially expressed in whole blood from patients with sepsis.

We probed published and public microarray datasets (1, 2) to discover the most significant gene expression changes in the blood of patients with sepsis. We identified significant differential expression of the heterogenous nuclear ribonucleoprotein hnRNP U in whole blood from patients with sepsis.

Telomere Function and the G-Quadruplex Formation are Regulated by hnRNP U

We examine the role of the heterogenous ribonucleoprotein U (hnRNP U) as a G-quadruplex binding protein in human cell lines. Hypothesizing that hnRNP U is associated with telomeres, we investigate what other telomere-related functions it may have. Telomeric G-quadruplexes have been fully characterized in vitro, but until now no clear evidence of their function or in vivo interactions with proteins has been revealed in mammalian cells. Techniques used were immunoprecipitation, DNA pull-down, binding assay, and Western blots. We identified hnRNP U as a G-quadruplex binding protein. Immunoprecipitations disclosed that endogenous hnRNP U associates with telomeres, and DNA pull-downs showed that the hnRNP U C-terminus specifically binds telomeric G-quadruplexes. We have compared the effect of telomere repeat containing RNA (TERRA) on binding between hnRNP U and telomeric (Tel) or single- stranded Tel (ssTel) oligonucleotides and found that ssTel binds stronger to TERRA than to Tel. We also show that hnRNP U prevents replication protein A (RPA) accumulation at telomeres, and the recognition of telomeric ends by hnRNP suggests that a G-quadruplex promoting protein regulates its accessibility. Thus, hnRNP U-mediated formation has important functions for telomere biology.

hnRNP U and DDX47 Are Novel FANCD2 Interactors That May Help to Resolve R-Loops during Mild Replication Stress

Background: Fanconi anemia proteins, encoded by at least 22genes (FANCA-W), constitute the Interstrand Cross Link (ICL) repair pathway. While FANCD2 is a master regulator of ICL repair, it accumulates at common fragile sites (CFS) during mild replication stress stimulated by low-dose Aphidicolin (APH) treatment. A recent study indicated that FANCD2 is required for efficient genome replication across the CFS regions. FANCD2 is also implicated in the regulation of R-loops levels. R-loops, which consist of DNA: RNA hybrids and displaced single-stranded DNA, are physiologically relevant in the genome and associate with immunoglobulin class switching, replication of mitochondrial DNA as well as transcriptional promoters or terminators. However, in any case, untimely formation of R-loops is a major threat to genome instability. Furthermore, it has been reported that R-loops which are induced by common slicing factor mutations in cases with myelodysplastic syndrome are linked to compromised proliferation of hematopoietic progenitors. It is also interesting to note that a recent study shows an interaction of FANCD2 with splicing factor 3B1 (SF3B1) and proposes their role in organizing chromatin domains to ensure coordination of replication and co-transcriptional processes. Methods: To examine the genome-wide distribution of FANCD2 protein, we set out to create a derivative of human osteosarcoma cell line, U2OS, which incorporated a 3×FLAG tag into the FANCD2 termination codon by genome editing. We performed chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis, and provide a genome-wide landscape of replication stress response involving FANCD2 in this cell line. Moreover, we purified the FANCD2 complex and analyzed by liquid chromatography-tandem mass spectrometry, and confirmed this interaction by co-immunoprecipitation (Co-IP) and proximal ligation assay (PLA) with FANCD2-3xFLAG. R-loops levels were assayed as the number of S9.6 (anti DNA:RNA hybrid antibody) stained foci per nucleus. Results: FANCD2 accumulation mostly occurs in the central portion of large transcribed genes, including CFS, and its accumulation appeared to be dependent on R-loop formation induced by transcription-replication collisions during mild replication stress. Moreover, our mass spectrometry analysis identified that FANCD2 interacts with several RNA processing factors including heterogeneous nucleoprotein U (hnRNP U), or DEAD box protein 47 (DDX47). We confirmed the interaction of these factors with FANCD2 by Co-IP as well as PLA. It was previously reported that defects in RNA-processing factors result in R-loop accumulation associated genome instability. Indeed, we found that treatment with siRNA against hnRNP U or DDX47 resulted in the increased number of the S9.6 foci. Furthermore, FANCD2 and hnRNP U or DDX47 appeared to function in an epistatic manner in suppressing APH-induced transcription-replication collisions as detected by PLA between PCNA and RNA polymerase II. Conclusion: We suggest that FANCD2 protects genome stability by recruiting RNA processing enzymes, including hnRNP U or DDX47, to resolve or prevent accumulation of R-loops induced by transcription-replication collisions during mild replication stress. Thus, our study may provide a novel insight to understand the mechanism of bone marrow failure and leukemogenesis in Fanconi anemia patients. Disclosures Takaori-Kondo: Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria.