Dasa Longman

Functional characterization of SR and SR-related genes in Caenorhabditis elegans

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR‐related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.

Multiple roles of the SR protein family in splicing regulation

The serine and arginine-rich proteins (SR proteins) are a highly conserved family of essential pre-mRNA splicing factors. SR proteins have a modular domain structure consisting of RNA binding and protein-protein interaction modules. SR proteins function as molecular adapters, mediating interactions between the pre-mRNA and the assembling spliceosome. Unlike most essential splicing factors, SR proteins have acquired an inherent flexibility that allows them to function at numerous steps in spliceosome assembly and therefore, to play key roles in regulation of splice site selection. In the postgenomics era it is widely accepted that alternative splicing of pre-mRNAs may significantly expand the capacity of the genome to generate the functional complexity of the proteome. Therefore, it is essential to understand both the mechanisms of splice site selection and how trans-acting factors, such as the SR proteins, are regulated. Within this chapter we will discuss how the structure of SR proteins influences their roles in alternative splice site selection.

Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of C. elegans

BACKGROUND SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs. RESULTS In the present study, we show that SRm160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleotide sequence. This suggests that it functions as a general coactivator by interacting with different SR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy. In the present study, we demonstrate that RNAi of CeSRm160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother. CONCLUSIONS The observation that simultaneous suppression of CeSRm160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.

DHX34 and NBAS form part of an autoregulatory NMD circuit that regulates endogenous RNA targets in human cells, zebrafish and Caenorhabditis elegans

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons but also regulates the abundance of cellular RNAs. We sought to identify transcripts that are regulated by two novel NMD factors, DHX34 and neuroblastoma amplified sequence (NBAS), which were identified in a genome-wide RNA interference screen in Caenorhabditis elegans and later shown to mediate NMD in vertebrates. We performed microarray expression profile analysis in human cells, zebrafish embryos and C. elegans that were individually depleted of these factors. Our analysis revealed that a significant proportion of genes are co-regulated by DHX34, NBAS and core NMD factors in these three organisms. Further analysis indicates that NMD modulates cellular stress response pathways and membrane trafficking across species. Interestingly, transcripts encoding different NMD factors were sensitive to DHX34 and NBAS depletion, suggesting that these factors participate in a conserved NMD negative feedback regulatory loop, as was recently described for core NMD factors. In summary, we find that DHX34 and NBAS act in concert with core NMD factors to co-regulate a large number of endogenous RNA targets. Furthermore, the conservation of a mechanism to tightly control NMD homeostasis across different species highlights the importance of the NMD response in the control of gene expression.

An evolutionarily conserved role for SRm160 in 3'-end processing that functions independently of exon junction complex formation

SRm160 (the SR-related nuclear matrix protein of 160 kDa) functions as a splicing coactivator and 3′-end cleavage-stimulatory factor. It is also a component of the splicing-dependent exon-junction complex (EJC), which has been implicated in coupling of pre-mRNA splicing with mRNA turnover and mRNA export. We have investigated whether the association of SRm160 with the EJC is important for efficient 3′-end cleavage. The EJC components RNPS1, REF, UAP56, and Y14 interact with SRm160. However, when these factors were tethered to transcripts, only SRm160 and RNPS1 stimulated 3′-end cleavage. Whereas SRm160 stimulated cleavage to a similar extent in the presence or absence of an active intron, stimulation of 3′-end cleavage by tethered RNPS1 is dependent on an active intron. Assembly of an EJC adjacent to the cleavage and polyadenylation signal in vitro did not significantly affect cleavage efficiency. These results suggest that SRm160 stimulates cleavage independently of its association with EJC components and that the cleavage-stimulatory activity of RNPS1 may be an indirect consequence of its ability to stimulate splicing. Using RNA interference (RNAi) in Caenorhabditis elegans, we determined whether interactions between SRm160 and the cleavage machinery are important in a whole organism context. Simultaneous RNAi of SRm160 and the cleavage factor CstF-50 (Cleavage stimulation factor 50-kDa subunit) resulted in late embryonic developmental arrest. In contrast, RNAi of CstF-50 in combination with RNPS1 or REFs did not result in an apparent phenotype. Our combined results provide evidence for an evolutionarily conserved interaction between SRm160 and the 3′-end cleavage machinery that functions independently of EJC formation.

Proteomic Analysis of SRm160-containing Complexes Reveals a Conserved Association with Cohesin

In this study, we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes and apply it to the characterization of complexes containing the SRm160 (serine/arginine repeat-related nuclear matrix protein of 160 kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3′-end processing via its N-terminal PWI nucleic acid-binding domain and is found in a post-splicing exon junction complex that has been implicated in coupling splicing with mRNA turnover, export, and translation. Consistent with these known functional associations, we found that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits SMC1α, SMC3, RAD21, and SA2. Gradient fractionation suggested that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and co-localization experiments, as well as combinatorial RNA interference in Caenorhabditis elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.

Identification and characterization of novel factors that act in the nonsense-mediated mRNA decay pathway in nematodes, flies and mammals

Nonsense‐mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome‐wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp‐1) and SEC13 (npp‐20), are also required for NMD in human cells. We also show that the C. elegans gene noah‐2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process.

Identification and characterization of RED120: A conserved PWI domain protein with links to splicing and 3′-end formation

Precursor (pre)‐mRNA splicing can impact the efficiency of coupled steps in gene expression. SRm160 (SR‐related nuclear matrix protein of 160 kDa), is a splicing coactivator that also functions as a 3′‐end cleavage‐stimulatory factor. Here, we have identified an evolutionary‐conserved SRm160‐interacting protein, referred to as hRED120 (for human Arg/Glu/Asp‐rich protein of 120 kDa). hRED120 contains a conventional RNA recognition motif and, like SRm160, a PWI nucleic acid binding domain, suggesting that it has the potential to bridge different RNP complexes. Also, similar to SRm160, hRED120 associates with snRNP components, and remains associated with mRNA after splicing. Simultaneous suppression in Caenorhabditis elegans of the ortholog of hRED120 with the orthologs of splicing and 3′‐end processing factors results in aberrant growth or developmental defects. These results suggest that RED120 may function to couple splicing with mRNA 3′‐end formation.

RNA TURNOVER IN EUKARYOTES: ANALYSIS OF SPECIALIZED AND QUALITY CONTROL RNA DECAY PATHWAYS

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