The expression of protein-coding genes is a multi-step process that begins with transcription and RNA processing (capping, splicing and 3'end formation) in the nucleus followed by export of mature mRNA to the cytoplasm for translation. All of the steps in gene expression are coupled to one another via an extensive network of physical and functional interactions that remains to be understood. Defects in gene expression are a major cause of human diseases, and conversely, gene expression is one of the major cellular processes that can be harnessed to diagnose and treat human diseases. Thus, it is vital to achieve a detailed understanding of the gene expression pathway. The broad goal of this proposal is to identify the factors and determine the mechanisms for coupling among the transcription, splicing, and mRNA export machineries. The central goal of Specific Aim 1 is to determine how transcription is coupled to splicing. A current model proposes that early splicing factors (U1 snRNP and SR proteins) are co-transcriptionally recruited to pre-mRNA to initiate spliceosome assembly. The protein FUS, which associates with U1 snRNP, SR proteins, and RNA polymerase II, was identified as a strong candidate for coupling transcription to splicing. FUS also associates with the SMN complex, which functions in snRNP biogenesis. RNAi and biochemical work will be carried out to determine the roles of FUS in coupling transcription to splicing and in U1 snRNP biogenesis.
In Specific Aim 2, a major effort is planned to determine how mutations in FUS cause the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). SMN-containing nuclear Gems are deficient in FUS knockdown HeLa cells and in ALS patient fibroblasts with a FUS mutation. Work is planned to determine whether Gem deficiency is a general phenotype in ALS. Splicing and snRNP biogenesis assays will also be carried out using nuclear extracts prepared from FUS knockdown HeLa cells and nuclear extracts containing FUS with ALS- causing patient mutations. These assays will also be performed using extracts prepared from motor neurons differentiated from mouse embryonic stem cells containing human ALS-causing patient mutations. This work is anticipated to add valuable insights into the pathogenesis of ALS as well as potentially leading to diagnostic tools for this disease. Finally, the central goal of Specific Aim 3 is to understand the function of the TREX complex, which is a highly conserved machinery that functions in mRNA export. TREX is linked to the spliceosomal Prp19 complex, and work is planned to determine how these complexes function in the export of both naturally intronless and spliced mRNAs. A combination of RNAi, factor recruitment assays, and RNA- protein interaction studies will be used to determine how these complexes function. Important insights into the essential cellular process of mRNA export is anticipated from these studies.

Public Health Relevance

Narrative Deciphering the pathway of gene expression and understanding each of the factors that function in this pathway are of critical medical importance. A thorough understanding of gene expression will provide powerful new opportunities for generating rapid and effective methods for diagnosing human disease as well as the critical goal of discovering highly targeted treatments for disease. Towards these objectives, the broad goal of this proposal is to achieve a detailed understanding of the mechanisms and factors involved in expression of protein-coding genes, with particular emphasis on how the different steps in the pathway are physically and functionally coupled to one another.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM043375-22A1
Application #
8369135
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1990-07-01
Project End
2016-07-31
Budget Start
2012-08-15
Budget End
2013-07-31
Support Year
22
Fiscal Year
2012
Total Cost
$739,638
Indirect Cost
$302,090
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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Shi, Min; Zhang, Heng; Wang, Lantian et al. (2015) Premature Termination Codons Are Recognized in the Nucleus in A Reading-Frame Dependent Manner. Cell Discov 1:
Yu, Yong; Chi, Binkai; Xia, Wei et al. (2015) U1 snRNP is mislocalized in ALS patient fibroblasts bearing NLS mutations in FUS and is required for motor neuron outgrowth in zebrafish. Nucleic Acids Res 43:3208-18
Folco, Eric G; Reed, Robin (2014) In vitro systems for coupling RNAP II transcription to splicing and polyadenylation. Methods Mol Biol 1126:169-77
Chesi, Alessandra; Staahl, Brett T; Jovičić, Ana et al. (2013) Exome sequencing to identify de novo mutations in sporadic ALS trios. Nat Neurosci 16:851-5
Lei, Haixin; Zhai, Bo; Yin, Shanye et al. (2013) Evidence that a consensus element found in naturally intronless mRNAs promotes mRNA export. Nucleic Acids Res 41:2517-25
Di Gregorio, Eleonora; Bianchi, Federico T; Schiavi, Alfonso et al. (2013) A de novo X;8 translocation creates a PTK2-THOC2 gene fusion with THOC2 expression knockdown in a patient with psychomotor retardation and congenital cerebellar hypoplasia. J Med Genet 50:543-51
Yamazaki, Tomohiro; Chen, Shi; Yu, Yong et al. (2012) FUS-SMN protein interactions link the motor neuron diseases ALS and SMA. Cell Rep 2:799-806
Folco, Eric G; Lei, Haixin; Hsu, Jeanne L et al. (2012) Small-scale nuclear extracts for functional assays of gene-expression machineries. J Vis Exp :

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