Podcasts about mrnp

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.511998v1?rss=1 Authors: Zeng, Y., Staley, J. P. Abstract: To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal splicing substrates after they have engaged the spliceosome. Although nuclear quality control mechanisms have been proposed to retain immature mRNPs, evidence indicates that discarded splicing substrates, including lariat intermediates, do export to the cytoplasm, as indicated by their translation and degradation by cytoplasmic nucleases. However, the mechanism for exporting these species has remained unknown. By single molecule (sm) RNA FISH in budding yeast, we have directly observed the nuclear export of lariat intermediates. Further, by crosslinking, export reporter assays, and smRNA FISH, we have demonstrated that the export of lariat intermediates requires the general mRNA export receptor Mex67p and three of its mRNA export adapter proteins, Nab2p, Yra1p, and Nlp3, establishing that both mRNAs and lariat intermediates share the same export machinery. Unexpectedly, the export of lariat intermediates, but not mRNA, requires an interaction between Nab2p and Mlp1p, a nuclear basket component implicated in retaining immature mRNPs, including unspliced pre-mRNA, in the nucleus of budding yeast. Finally, the export of lariat intermediates, like mRNA, relies on the E3 ubiquitin ligase Tom1p and its target sites in Yra1p. Overall, our data indicate that the nuclear basket can promote, rather than antagonize, the export of an immature mRNP. Further, our data imply that the export of discarded lariat intermediates requires both Mlp1p-dependent docking onto the nuclear basket and subsequent Tom1p-mediated undocking, a mechanism our data suggests generalizes to the export of mRNA but in a manner obscured by redundant pathways. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.02.184796v1?rss=1 Authors: Singh, A., Hulsmeier, J., Kandi, A. R., Pothapragada, S. S., Hillebrand, J., Petrauskas, A., Agrawal, K., RT, K., Thiagarajan, D., Vijayraghavan, K., Ramaswami, M., Bakthavachalu, B. Abstract: Ataxin-2 is a conserved translational control protein associated with spinocerebellar ataxia type II (SCA2) and amyotrophic lateral sclerosis (ALS) as well as an important target for ALS therapeutics under development. Despite its clinical and biological significance, Ataxin-2's activities, mechanisms and functions are not well understood. While Drosophila Ataxin-2 (Atx2) mediates mRNP condensation via a C-terminal intrinsically disordered domain (cIDR), how Ataxin-2 IDRs work with structured (Lsm, Lsm-AD, and PAM2) domains to enable positive and negative regulation of target mRNAs remains unclear. Using TRIBE (Targets of RNA-Binding Proteins Identified by Editing) technology, we identified and analysed Atx-2 target mRNAs in the Drosophila brain. We show that Atx2 preferentially interacts with AU-rich elements (AREs) in 3'UTRs and plays a broad role in stabilization of identified target mRNAs. Strikingly, Atx2 interaction with its targets is dependent on the cIDR domain required for neuronal-granule formation. In contrast, Atx2 lacking its Lsm domain not only interacts more efficiently with the target mRNA identified, but also forms larger RNP granules. Providing an extensive dataset of Atx2-interacting brain mRNAs, our results demonstrate that Atx2: (a) interacts with target mRNAs within RNP granules; (b) modulates the turnover of these target mRNAs; (c) has an additional essential role outside of mRNP granules; and (d) contains distinct protein domains that drive or oppose RNP-granule assembly. These findings increase understanding of neuronal translational control mechanisms and inform Ataxin-2-based interventions in development for SCA2 and ALS. Copy rights belong to original authors. Visit the link for more info

For more information, please visit: http://bitesizebio.com/webinar/25961/deciphering-steps-of-mrnp-assembly-in-developing-oocytes-using-super-resolution-microscopy/ All mRNA molecules recruit specific proteins to form ribonucleoprotein complexes (mRNPs). Composition and localization of many mRNPs change dynamically from translation to decay. Microscopic techniques with high spatial and temporal resolution are invaluable for studying mRNP biogenesis. We have developed new tools based on fluorogenic forced intercalation (FIT) probes for RNA detection, quantification and interference in biological samples. The probes contain a thiazole orange (TO) dye introduced at a position normally occupied by a nucleobase. Upon binding to target nucleic acids, the TO dye increases its quantum yield and brightness substantially (greater than10 fold). These probes detect mRNA in a rapid, wash-free FISH setup using conventional wide-field microscopy. It is an ideal tool for RNA localization screens. Nuclease resistant FIT probes containing a locked nucleic acid adjacent to the TO dye are bright and contrasted enough for use in live imaging. These probes can also be designed to target functional elements of RNAs to test the role of those in RNP biogenesis. Absorption and emission spectra of TO are sufficiently different from EGFP to enable high sensitivity and specificity RNA-protein co-localization analysis, even with super-resolution, to study the RNA interactome. LNA modified FIT probes are excellent subjects for STED microscopy as duplex formation greatly increases their fluorescence lifetime.

Mon, 27 Jun 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13203/ https://edoc.ub.uni-muenchen.de/13203/1/Coordes_Britta.pdf Coordes, Britta ddc:540, ddc

The control of mRNA translation mediated by RNA-binding proteins (RBPs) is a key player in modulating gene expression. In S. cerevisiae, the multi-KH domain protein Scp160 associates with a large number of mRNAs and is present on membrane-bound and, to a lesser extent, cytosolic polysomes. Its binding site on the ribosome is close to the mRNA exit tunnel and in vicinity to Asc1, which constitutes a binding platform for signaling molecules. The present study focused on the closer characterization of the Scp160-ribosome interaction and on the suggested function of Scp160 in modulating the translation of specific target mRNAs. Using affinity purifications, the partial RNA-dependence of the Scp160-ribosome association was confirmed. In contrast to published results, ribosome association was found to be only slightly reduced but not abolished in the absence of Asc1 or the last two KH domains. Furthermore, the putative elongation regulator Stm1 was identified as a co-purifier of Scp160. In subcellular fractionation experiments, RNA-binding mutants of Scp160 were present in the ribosome-free cytosolic fraction and therefore partially deficient in ribosome association and/or mRNP formation. However, no physiological conditions were found that equally induce a shift of wildtype Scp160 towards the cytosolic fraction. Within the scope of a translational profiling approach, microarray analyses of RNA isolated from sucrose density gradient fractions were performed and led to the identification of a set of mRNAs that shift their position within the gradients upon Scp160 depletion, indicating changes in their translation rates. Consistent with the membrane localization of Scp160, transcripts encoding secreted proteins were significantly enriched. Using immunoprecipitation and subsequent quantitative real-time PCR (qRT-PCR), the interaction of Scp160 with a subgroup of the identified targets was confirmed and it was shown that their binding is dependent on the conserved GXXG motifs in the two C-terminal KH domains of Scp160. Furthermore, data were obtained indicating that Scp160 can act as a translational activator on some of its target mRNAs, probably on the level of translation elongation. Finally, first evidence was provided that the translational misregulation of specific target transcripts may be involved in the polyploidization that is a hallmark of Scp160-deprived cells. In summary, these data substantiate the assumption that Scp160 is involved in translational regulation of a specific, functionally related subset of mRNAs. This finding is in good accordance with the emerging view that RBPs co-regulate multiple transcripts in order to allow faster adaptation to environmental changes.

The hallmark of eukaryotic evolution was the development of the nucleus in cells. This compartmentalization requires the nucleocytoplasmic transport of thousands of molecules. The gate into and out of the nucleus is the nuclear pore complex (NPC). One of the molecules that needs to be exported from the nucleus is messenger RNA (mRNA). mRNA associates with proteins in the nucleus forming a messenger ribonucleoprotein particle (mRNP). mRNPs bind to dedicated transport factors that facilitate movement through the NPC. One protein that associates to mRNPs is the helicase Dbp5, which belongs to the DEAD-box family of RNA helicases. Dbp5 is essential for mRNA export in both yeast and humans. It binds RNA and is concentrated and locally activated at the cytoplasmic side of the nuclear pore complex, where it interacts with the cytoplasmic nucleoporin Nup214. In my PhD work, I have determined the crystal structures of human Dbp5 bound to RNA and AMPPNP, and bound to Nup214. I designed and performed in vitro assays, which show that binding of Dbp5 to nucleic acid and to Nup214 is mutually exclusive. The interactions are mediated by conserved residues, implying a conserved recognition mechanism. These results suggest a framework for the consecutive steps leading to the release of mRNA at the final stages of nuclear export. More generally, they provide a paradigm for how binding of regulators can specifically inhibit DEAD-box proteins.

Wed, 8 Jul 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11091/ https://edoc.ub.uni-muenchen.de/11091/1/Mueller_Marisa.pdf Müller, Marisa ddc:540, ddc:500, Fakultät für Chemie und Pha

Gene expression is highly regulated and interconnected to processes like mRNP processing, mRNA export as well as to DNA repair and replication. The first step of gene expression is the transcription of protein coding genes by RNA polymerase II. Transcription is controlled by general transcription factors, the phosphorylation of the C-terminal domain of Rpb1, the largest subunit of RNA polymerase II, and chromatin modifications that allow proper accessibility of the DNA. A major player in these coupling processes is the TREX complex, coupling transcription elongation to the nucleo-cytoplasmic export of the mRNP via the nuclear pore complex. Particularly, the THO subcomplex of TREX has functions in hyperrecombination, nucleotide excision repair and transcription coupled repair. A genetic screen with TREX components, performed to identify genes involved in these processes, lead to the identification of the cyclin dependent kinase Bur1. Bur1 and its cyclin Bur2 are needed for efficient transcription elongation by RNA polymerase II by regulating the methylation of histone tails. Interestingly, Bur1 interacts in vivo with RPA, a single strand DNA binding protein essential for genome stability. This biochemical interaction raised the idea of a novel interconnection between transcription, chromatin modification and genome maintenance. Mutations in the BUR1 as well as in the RFA1 gene lead to sensitivity to drugs that cause DNA damage and replication or transcription stress. Deletion of the C-terminus of Bur1, which is sufficient for the binding to RPA, also renders cells sensitive to those agents. This shows the functional significance of this protein-protein interaction in the cell upon stress induction. However, attempts to identify the DNA repair pathway Bur1 is involved in showed that mutations in BUR1 do not behave epistatic with deletions of specific pathways. This result points to a more general, maybe regulatory role of Bur1 in the response to DNA damage. It is interesting to note that mutations in BUR1 lead to increased genomic instability as they show the appearance of a higher amount and longer persistence of nuclear foci, DNA repair “factories” that contain, among other proteins, Rfa1 and Rad52. Furthermore, RFA1 mutants show decreased levels of histone H3 alone as well as lower levels of histone H3 Lysine 4 trimethylation, a mark for transcription elongation, when combined with a mutation in BUR1. The RFA1 mutant is also impaired in the expression of a β-galactosidase reporter gene, pointing to a function of RPA in transcription. Interestingly, combining BUR1 and RFA1 mutants leads to a lower susceptibility of cells to stress than one of the mutations alone. On the one hand, this could be elucidated by better growth of the double mutant strains upon stress compared to the single mutants. On the other hand, whole genome expression analysis shows that the double mutant strain clusters with the bur1 mutant whereas the rfa1 mutant does not, showing that its expression pattern is closer to the bur1 mutant. Both results show that the protein complexes have antagonistic roles as the combination of both mutations leads to a suppression phenotype based on differential gene expression. Taken together, a function of Bur1 in genome maintenance could be established, as well as an effect of RPA on transcription elongation and chromatin modification. The results provide a possibility to speculate about a coupling of transcription and genome stability mediated by the interaction of Bur1-2 with RPA.

Gene expression encompasses a multitude of different steps, starting with transcription in the nucleus, co-transcriptional processing and packaging of the mRNA into a mature mRNP, export of the mRNP through the nuclear pore and finally the translation of the message in the cytoplasm. The central coordinator for coupling of the nuclear events is the differentially phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAP II). The phosphorylation pattern of the CTD not only dictates the progression through the transcription cycle but also the recruitment of mRNA processing machineries. Coupling of transcription to mRNA export is achieved by the TREX complex, which consists in the yeast S. cerevisiae of the heterotetrameric THO complex important for transcription elongation, the SR-like proteins Gbp2 and Hrb1, and Tex1 and the mRNA export factors Sub2 and Yra1. By direct interaction with Yra1, the mRNA export receptor Mex67-Mtr2 is then recruited to the mRNP and transports the mRNP through the nuclear pore complex to the cytoplasm. In a genetic screen for factors that are crucial for TREX complex function in the living cell, Ctk1, a cyclin dependent kinase (CDK) that phosphorylates the C-terminal domain (CTD) of RNAP II during transcription elongation, was identified (Hurt et al. 2004). Surprisingly, besides the TREX components Gbp2 and Hrb1, Ctk1 co-purified ribosomal proteins and translation factors. Using sucrose density centrifugation, it could be shown that Ctk1 indeed associates with translating ribosomes in vivo, suggesting a novel function of this protein in translation. This assumption was confirmed by in vitro translation assays: loss of Ctk1 function leads to a reduction in translational activity. More specifically, Ctk1 is important for efficient translation elongation and contributes to the accurate decoding of the message. Cells depleted for Ctk1 are more sensitive towards drugs that impair translational accuracy and show an increase in the frequency of miscoding in vivo. The function of Ctk1 during decoding of the message is most likely direct, as in extracts of cells depleted for Ctk1 the defect in correct decoding of the message can be restored to wild type levels by addition of purified CTDK-I complex. An explanation for the molecular mechanism of Ctk1’s function is provided by the identification of Rps2 as a novel substrate of Ctk1. Rps2 is a protein of the small ribosomal subunit, located at the mRNA entry tunnel and known to be essential for translational accuracy. Importantly, Rps2 is phosphorylated on serine 238 by Ctk1, and cells containing an rps2-S238A mutation show an increased sensitivity towards drugs that affect translational accuracy and an increase in miscoding as determined by in vitro translation extracts. The role of Ctk1 in translation is probably conserved as CDK9, the mammalian homologue of Ctk1, also associates with polysomes. Since Ctk1 interacts with the TREX complex, which functions at the interface of transcription and mRNA export, Ctk1 might bind to the mRNP during transcription and accompany the mRNP to the ribosomes, where Ctk1 enhances efficient and accurate translation of the mRNA. This study could be an example of a novel layer of control in gene expression: the composition of the mRNP determines its translational fate, including efficiency and accuracy of translation.

Fragile X syndrome is the most frequent form of heritable mental retardation. In the majority of cases the disease is caused by transcriptional silencing of the FMR1 gene in response to the expansion of CGG repeats in the 5´UTR of the gene, leading to a lack of fragile X mental retardation protein (FMRP). Only a few patients exhibit missense-mutations in the coding region of FMR1 leading to an aberrant gene product. However, little is known about how FMRP deficiency or malfunction leads to the pathophysiology of Fragile X syndrome. FMRP is an RNA binding protein that associates with translating polyribosomes as part of a large messenger ribonucleoprotein (mRNP). In the present study, it was investigated whether the protein can act as a regulator of translation. Using recombinant FMRP, it was shown that the protein suppresses translation of different mRNAs in rabbit reticulocyte lysate. Interestingly, FMRP containing an amino acid substitution at position 304 (FMRPI304N), originally identified in a severely affected patient, renders the protein incapable of interfering with translation. In vitro binding experiments revealed that FMRPI304N, in contrast to wildtype FMRP, was incapable of forming homo-oligomers. However, its affinity for mRNA was not altered. These results suggest that oligomerization is a prerequisite for the function of FMRP in translational inhibition. In order to identify the protein region responsible for homo- and hetero-oligomerization with the two autosomal homologues FXR1 and FXR2, an array of FMRP deletion mutants were tested for oligomerization properties. In vitro binding experiments showed that a putative coiled-coil domain (aminoacids 112 to 215) was required for mutual interaction of all three proteins. Finally, it was examined whether FMRP is post-translationally modified and whether this may play a role in the function of this protein. It was initially demonstrated that both, mammalian cell extract and casein-kinase II, are capable of FMRP phosphorylation in vitro. Furthermore phospho-aminoacid analysis of immunoprecipitated human FMRP revealed phosphorylation of serine residues. Additionally, the protein can be methylated in vitro in the C-terminal part. The outlined experimental strategy may be utilized as a tool for the identification and characterization of FMRP-modifying enzymes. The obtained data identify FMRP as a negative regulator of translation and suggest that misregulation of translation of specific mRNAs lead to the disease phenotype. Analysis of the biogenesis of spliceosomal U-snRNP was the second project of the Ph.D. thesis. This process involves the transient export of nuclear encoded U snRNA to the cytoplasm, the assembly with a set of spliceosomal proteins (termed Sm-proteins B/B’, D1, D2, D3, E, F, and G) and the nuclear import of the assembled particle to the nucleus. The assembly reaction of U-snRNP, although a spontaneous process in vitro, is facilitated in vivo by a large number of proteins, including SMN, the protein mutated in the neuromuscular disorder spinal muscular atrophy. These factors are organised in two functional units, termed PRMT5-complex and SMN-complex, that successively cooperate in the assembly reaction. In a first step, the PRMT5-complex sequesters newly synthesized Sm proteins and converts arginines in SmB, D1 and D3 to symmetrical dimethylarginines (sDMA). This enhances their transfer to the SMN-complex, which facilitates the assembly reaction with the U snRNA. Whereas in vitro methylation experiments have identified PRMT5 as the catalytic component of the PRMT5 complex, the functions of the two known cofactors of this enzyme, pICln and WD45, were unknown. Using a biochemical approach, an interaction map of the PRMT5 complex has been established. These studies revealed that oligomeric PRMT5 directly interacts with pICln and WD45 via distinct domains. Interestingly, in vitro methylation experiments further indicated that both cofactors strongly activate the catalytic activity of the methyltransferase. The transfer of modified Sm proteins to the SMN-complex was studied in a newly established assay system. Sm proteins form stable heterooligomeric complexes composed of SmB/D3, SmD1/D2 and SmE/F/G and it is believed that these complexes are intermediates in the assembly reaction. Quite unexpectedly, the studies presented in this thesis indicate that Sm proteins bind initially as monomers to the PRMT5-complex to allow for the efficient methylation and transfer to the SMN complex. The data suggest that hetero oligomerization takes place in a late step of the assembly reaction, possibly at the SMN complex prior to U snRNP assembly.