Operons are an important feature of the C. elegans genome. Their transcripts are polycistronic pre-mRNAs that are processed by 3'end formation and trans-splicing. These two events occur in close proximity between operon genes. Although 3'end formation is generally accompanied by transcription termination, in operons it is not. We have identified the key sequences and many of the key trans-acting proteins and snRNAs responsible for carrying out these events, and we are determining what roles they play and how. The key sequence required for both the trans-splicing and for preventing transcription termination is the Ur element that occurs ~50 bp downstream of the 3'end cleavage site. We will determine what binds there. We have recently succeeded in obtaining several templates from operons that are correctly trans-spliced in vitro in a C. elegans embryo extract. This indicates that correct trans-splicing is a property of proteins that bind to the RNA, rather than requiring co-transcriptional RNA processing. We will exploit the in vitro system to test a wide range of mutant substrates to identify all sequences required for correct trans-splicing. We will then identify the macromolecules that act at these sequences and determine what roles they play. Normally transcription termination accompanies 3'end formation, but in operons this cannot be the case. We will use chromatin IP to analyze what phosphorylation events occur to the RNA polymerase CTD as it traverses an operon. These studies should provide important new insight into how 3'end cleavage can occur without accompanying transcription termination in operons. This work could also provide strong support for, or refutation of, the torpedo model for transcription termination. We are also studying the roles of the protein components of the trans-splicing snRNPs. Functional studies involving mutants of these components and how they interact with mutants in functionally related genes are proposed. We have discovered that two of these proteins are bound to a novel snRNA, Sm Y, currently the only snRNA whose function is not known. We will study the possible role of this RNA in trans-splicing and operon pre-mRNA processing. We have hypothesized that these proteins are required for recycling the Sm proteins from the branched intermediates following trans-splicing, and we will test this model using both in vivo and in vitro experiments. Finally, we have devised a genetic screen for mutants in genes required for proper trans-splicing.

Public Health Relevance

This project focuses on basic means of gene expression in nematodes, organisms that are responsible for a huge disease burden in humans, as well as domestic animals and cultivated plants. The process under study here, trans-splicing, occurs in nematodes but not their hosts. It is hoped that if we can come to understand this process in detail it may suggest ways to interfere with it, such that the nematode would be sensitive to a treatment that the host is not. Besides this, the basic knowledge we obtain from this project may be important in understanding mechanisms of gene expression in general, especially processing of mRNAs, and this basic knowledge may provide clues for means to treat mRNA splicing defects responsible for many genetic diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042432-19
Application #
8135028
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
1992-05-01
Project End
2013-08-31
Budget Start
2011-09-01
Budget End
2013-08-31
Support Year
19
Fiscal Year
2011
Total Cost
$355,313
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
Saldi, Tassa K; Ash, Peter Ea; Wilson, Gavin et al. (2014) TDP-1, the Caenorhabditis elegans ortholog of TDP-43, limits the accumulation of double-stranded RNA. EMBO J 33:2947-66
Saito, Taro Leo; Hashimoto, Shin-ichi; Gu, Sam Guoping et al. (2013) The transcription start site landscape of C. elegans. Genome Res 23:1348-61
Blumenthal, Thomas (2012) Trans-splicing and operons in C. elegans. WormBook :1-11
Morton, J Jason; Blumenthal, Thomas (2011) Identification of transcription start sites of trans-spliced genes: uncovering unusual operon arrangements. RNA 17:327-37
Allen, Mary Ann; Hillier, LaDeana W; Waterston, Robert H et al. (2011) A global analysis of C. elegans trans-splicing. Genome Res 21:255-64
Lasda, Erika L; Kuersten, Scott; Blumenthal, Thomas (2011) SL trans-splicing in a Caenorhabditis elegans in vitro extract. Cold Spring Harb Protoc 2011:pdb.prot5574
Lasda, Erika L; Allen, Mary Ann; Blumenthal, Thomas (2010) Polycistronic pre-mRNA processing in vitro: snRNP and pre-mRNA role reversal in trans-splicing. Genes Dev 24:1645-58
Garrido-Lecca, Alfonso; Blumenthal, Thomas (2010) RNA polymerase II C-terminal domain phosphorylation patterns in Caenorhabditis elegans operons, polycistronic gene clusters with only one promoter. Mol Cell Biol 30:3887-93
MacMorris, Margaret; Kumar, Madhur; Lasda, Erika et al. (2007) A novel family of C. elegans snRNPs contains proteins associated with trans-splicing. RNA 13:511-20
Liu, Yingmiao; Kuersten, Scott; Huang, Tao et al. (2003) An uncapped RNA suggests a model for Caenorhabditis elegans polycistronic pre-mRNA processing. RNA 9:677-87

Showing the most recent 10 out of 25 publications