The discovery that many intron-containing genes can be cotranscriptionally spliced has led to an increased understanding of how splicing and transcription are intricately intertwined. ...Cotranscriptional splicing has been demonstrated in a number of different organisms and has been shown to play roles in coordinating both constitutive and alternative splicing. The nature of cotranscriptional splicing suggests that changes in transcription can dramatically affect splicing, and new evidence suggests that splicing can, in turn, influence transcription. In this chapter, we discuss the mechanisms and consequences of cotranscriptional splicing and introduce some of the tools used to measure this process.
Assembly of the spliceosome onto pre-mRNA is a dynamic process involving the ordered exchange of snRNPs to form the catalytically active spliceosome. These ordered rearrangements have recently been ...shown to occur cotranscriptionally, while the RNA polymerase is still actively engaged with the chromatin template. We previously demonstrated that the histone acetyltransferase Gcn5 is required for U2 snRNP association with the branchpoint. Here we provide evidence that histone acetylation and deacetylation facilitate proper cotranscriptional association of spliceosomal snRNPs. As with GCN5, mutation or deletion of Gcn5-targeted histone H3 residues leads to synthetic lethality when combined with deletion of the genes encoding the U2 snRNP components Lea1 or Msl1. Gcn5 associates throughout intron-containing genes and, in the absence of the histone deacetylases Hos3 and Hos2, enhanced histone H3 acetylation is observed throughout the body of genes. Deletion of histone deacetylaces also results in persistent association of the U2 snRNP and a severe defect in the association of downstream factors. These studies show that cotranscriptional spliceosome rearrangements are driven by dynamic changes in the acetylation state of histones and provide a model whereby yeast spliceosome assembly is tightly coupled to histone modification.
U1 snRNA Rewrites the “Script” Merkhofer, Evan C.; Johnson, Tracy L.
Cell,
07/2012, Letnik:
150, Številka:
1
Journal Article
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Odprti dostop
Expression of eukaryotic mRNAs requires the collaboration of a host of RNA processing factors acting upon the transcript. Berg et al. describe how a pre-mRNA splicing factor modulates the activity of ...the polyadenylation machinery to regulate mRNA length, with important implications for isoform expression in activated neuronal and immune cells.
The membrane bound receptor tyrosine kinase Her2 is overexpressed in approximately 30% of human breast cancers, which correlates with poor prognosis. Her2-induced signaling pathways include MAPK and ...PI3K/ Akt, of which the latter has been shown to be critical for Her2.sup.+ breast cancer cell growth and survival. In addition, the NF-κB pathway has been shown to be activated downstream of Her2 overexpression; however, the mechanisms leading to this activation are not currently clear. Using Her2.sup.+/ER.sup.- breast cancer cells, we show that Her2 activates NF-κB through the canonical pathway which, surprisingly, involves IKKα. Knockdown of IKKα led to a significant decrease in transcription levels of multiple NF-κB-regulated cytokine and chemokine genes. siRNA-mediated knockdown of IKKα resulted in a decrease in cancer cell invasion, but had no effect on cell proliferation. Inhibition of the PI3K/Akt pathway had no effect on NF-κB activation, but significantly inhibited cell proliferation. Our study suggests different roles for the NF-κB and PI3K pathways downstream of Her2, leading to changes in invasion and proliferation of breast cancer cells. In addition this work indicates the importance of IKKα as a mediator of Her2-induced tumor progression. Oncogene (2010) 29, 1238-1248; doi: 10.1038/onc.2009.410; published online 30 November 2009 Keywords: Her2; IKKalpha; NF-KappaB
Regulation of gene expression occurs at multiple levels, including the removal of non‐coding sequences from protein‐coding pre‐messenger RNA transcripts, commonly known as pre‐mRNA splicing. Correct ...gene expression requires high fidelity of pre‐mRNA splicing, as up to 15% of mutations leading to disease may be a result of aberrant pre‐mRNA splicing, including cystic fibrosis, spinal muscular atrophy (SMA) and some leukemias. Pre‐mRNA splicing is a multistep process that requires several proteins and RNAs for ATP hydrolysis, including multiple essential DEAD‐box proteins such as Prp5. Here we investigate the role of the DEAD‐box protein Prp5 in splicing using the yeast Saccharomyces cerevisiae as a model. Prp5 has previously been shown to be required for ATP‐dependent RNA remodeling in splicing, facilitating binding of the U2 snRNA to the branchpoint sequence in the pre‐mRNA intron. In addition, unpublished research has revealed genetic interactions between Prp5 and non‐splicing genes such as Gcn5 and Spt8. In this study, additional potential genetic interactions involving Prp5 were investigated. To identify these interactions, a transposon mutagenesis screen was employed. Approximately 20,000 mutations were screened, 61 of which showed putative genetic suppression of the temperature sensitive prp5‐1 mutation. Subsequent growth assays confirmed that many of these mutations exhibited genetic suppression, indicating important genetic interactions between these genes and Prp5. hiTAIL PCR was utilized to identify a number of these genetic interactions, including an interaction between Prp5 and the MMO1 gene, a verified protein in S. cerevisiae with unknown function. Additional experiments to characterize this interaction with MMO1 are currently being performed. Furthermore, we have used high‐throughput sequencing to identify an additional subset of the interactions from the transposon screen. This study will help shed light upon the role of the DEAD‐box protein Prp5 in pre‐mRNA splicing.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract only
The removal of non‐coding sequences from protein‐coding pre‐messenger RNAs is required for proper gene expression. This highly conserved process, commonly known as RNA splicing, is ...mediated by a large ribonucleoprotein complex known as the spliceosome. Dysregulation of RNA splicing is common in disease, as up to 15% of mutations leading to disease have been attributed to an effect on pre‐mRNA splicing. The DExD/H‐box family of ATPases, including Prp5, play a critical role in mediating ATP‐dependent RNA remodeling in splicing. One pressing question that remains is what factors are required for rapid ATP‐dependent rearrangements of the spliceosome that underlie high fidelity splice site recognition. Using
Saccharomyces cerevisiae
, our studies of the mechanisms of spliceosomal rearrangements have identified striking genetic interactions between the Rpn10 subunit of the proteasome and the ATP‐dependent spliceosomal RNPase Prp5. Deletion of
rpn10
rescues the slow growth phenotype exhibited by strains containing
prp5
ATPase mutations, and leads to an increase in splicing
in vitro
. Furthermore, we show that Rpn10 can regulate protein levels of multiple spliceosome components, including Cus2, and
rpn10
shows strong genetic interactions with this gene. These intriguing results suggest a novel role of the regulatory subunit of the proteasome in mediating rearrangements between components of the spliceosome.
The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also ...pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Overexpression of the membrane-bound receptor tyrosine kinase Her2 (ErbB-2, EGFR2) occurs in approximately 30% of all breast cancers and typically correlates with poor prognosis. Overexpression of ...Her2 leads to activation of multiple downstream pathways including the MAPK, PI3K/Akt and NF-κB pathways. Her2 has been previously reported to activate the NF-κB pathway. However, the mechanism by which this occurs is poorly elucidated. In this report, we utilize an siRNA approach to investigate the role that the different Inhibitor of Kappa-B Kinase (IKK) subunits play in activation of NF-κB downstream of Her2. We show that IKKα plays a previously unreported role in NF-κB activation via the canonical pathway in Her2-overexpressing breast cancer cells. Furthermore, IKKα plays an important role in NF-κB regulated gene expression and induction of an invasive phenotype in these cells, independent of PI3K. This activation of NF-κB by Her2 also requires the NF-κB pathway kinase, TGF-β-activated kinase 1 (TAK1). Finally, we also show that inhibition of IKKα by siRNA leads to activation of SPARC, a member of the BM-40 family of genes which are often dysregulated in cancer. Activation of the NF-κB pathway can directly suppress expression of SPARC through a mechanism at the promoter of SPARC, proximal to the transcriptional start site. In summary, we present data elucidating the role and consequences of NF-κB activation in Her2-overexpressing breast cancer.
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