Project description:Long-range mRNA transport is crucial for the spatio-temporal regulation of gene expression, and its malfunction is linked to neurological disorders. The pentameric FERRY Rab5 effector complex is the molecular link between mRNA and the early endosome in mRNA intracellular distribution. Here, we determine the cryo-EM structure of the human FERRY complex, composed of Fy-1 to Fy-5. The structure reveals a clamp-like architecture, in which two arm-like appendages, each consisting of Fy-2 and a Fy-5 dimer, protrude from the central Fy-4 dimer. We demonstrate that the coiled-coil domains of Fy-2 are flexible and project into opposite directions from the FERRY complex core. While the C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils together with Fy-5 bind mRNA. Thus, Fy-2 serves as binding hub that connects not only all five complex subunits, but also mediates the binding to mRNA and to the early endosome via Rab5. The FERRY structure provides novel mechanistic insight into long-distance mRNA transport.
Project description:The eukaryotic microrchidia (MORC) protein family are DNA gyrase, Hsp90, histidine kinase, MutL (GHKL)-type ATPases involved in gene expression regulation and chromatin compaction. The molecular mechanisms underlying these activities are incompletely understood. Here, we studied the full-length human MORC2 protein biochemically. We identified a DNA binding site in the C-terminus of the protein, and we observe that this region is heavily phosphorylated in cells. Phosphorylation of MORC2 appears to reduce its affinity for DNA and exclude the protein from the nucleus. We observe that DNA binding by MORC2 reduces its ATPase activity and that MORC2 can topologically entrap multiple DNA substrates between its N-terminal GHKL and C-terminal coiled coil 3 dimerization domains. Finally, we observe that the MORC2 C-terminal DNA binding region is required for gene silencing in cells. Together, our data provide a model to understand how MORC2 engages with DNA substrates to mediate gene silencing.
Project description:MMARY THUMPD2 installs N2-methylguanosine (m2G) at position 72 of the U6 snRNA, and this modification, which lies at the catalytic core of the spliceosome, is required for efficient splicing of weak splice sites. However, when during the U6 lifecycle this modification is installed and what features of the U6 snRNA and THUMPD2 are required for methylation of G72 remain elusive. Here, we show that U6 associated with THUMPD2 is not oligouridylated and that late-acting U6 snRNP biogenesis factors are enriched with THUMPD2. We map RNA crosslinking sites within the N-terminal region and C-terminal Rossman-fold methyltransferase domain of THUMPD2, and show the requirement of these domains for THUMPD2 interaction with U6 in cells. Using a newly developed m2G-sensitive deoxyribozyme to monitor U6-m2G72 levels in cellular RNAs, we demonstrate the requirement of SAM binding and interaction with the THUMPD2 cofactor TRMT112 for efficient methylation. Our data further reveal that 2’-O-methylation of C62, C63 and A70 do not increase the affinity of THUMPD2 for the U6 internal stem-loop (ISL), but enhance m2G methylation of G72. Nucleotide substitutions within the loop region of the ISL that extend the stem similarly increase m2G72 installation, demonstrating that the stability of the ISL is key for methylation by THUMPD2. Together these results provide a new layer of understanding of how an RNA modification that fine-tunes pre-mRNA splicing is introduced.
Project description:Proteins interacting with mRNA in mouse macrophages were identified by crosslinking and oligodT capture. Differential interaction was investigated in macrophages before and after treatment with LPS.
Project description:RNA-binding proteins are increasingly recognized as regulatory component of post-transcriptional gene expression. RBPs interact with mRNAs via RNA-binding domains and these interactions affect RNA availability for translation, RNA stability and turn-over thus affecting both RNA and protein expression essential for developmental and stimulus specific responses. Here we investigate the effect of severe drought stress on the RNA-binding proteome to gain insights into the mechanisms that govern drought stress responses at the systems level
Project description:RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR-association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in poly-pyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing. HuR (ELAVL1) PAR-CLIP
Project description:We reveal that the GNL3 paralogue GTPase, GNL3L, associates with pre-ribosomes. We map RNA contact sites on GNL3L as well as GNL3L binding sites on pre-rRNAs. Lack of GNL3L impairs synthesis of the 60S rRNAs and expression of GTPase inactive GNL3L causes defects in early steps of pre-rRNA processing. Impaired GTP hydrolysis by GNL3L leads to its accumulation on pre-60S particles, together with other assembly factors with proximal binding sites.
Project description:RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.
Project description:Alpha synuclein in it's native state in solution was crosslinked using a variety of short-range crosslinking reagents. Crosslinks detected by mass spectrometry were then used as constraints in a discrete molecular dynamics simulation of the synuclein protein, and a structural ensemble of the protein was determined.
Project description:High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute protein members can pinpoint microRNA target sites within tens of bases, but leaves the identity of the microRNA unresolved. A flexible computational framework that integrates sequence with cross-linking features reliably identifies the microRNA family involved in each binding event, considerably outperforms sequence-only approaches, and quantifies the prevalence of noncanonical binding modes. Ago2 (Argonaute 2) PAR-CLIP and RNA deep sequencing of Epstein-Barr virus B95.8-infected Lymphoblastoid Cell Lines (LCLs)