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N Daniels Dissertation.pdf (20.61 MB)

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Functional Analyses of Human DDX41 and LUC7-like Proteins Involved in Splicing Regulation and Myeloid Neoplasms

Daniels, Noah James
http://orcid.org/0000-0002-1688-0933
http://rave.ohiolink.edu/etdc/view?acc_num=case1648820426769147

Abstract Details

2022, Doctor of Philosophy, Case Western Reserve University, Molecular Medicine.
Myeloid neoplasms consist of a large group of hematological malignancies frequently characterized by genetic abnormalities in splicing factors. We investigated the role of several mammalian splicing factors, DDX41 and members of the LUC7-like family, in splicing and disease pathogenesis. DDX41 encodes a DEAD-box RNA helicase with functions implicated in innate immunity, snoRNA processing, and pre-mRNA splicing. Germline DDX41 mutations predispose to late-onset myeloid neoplasms with somatic mutations commonly occurring on the second allele. We designed a system to deplete DDX41 in human and mouse cell lines for in vitro mechanistic studies. However, technical problems arose, preventing utilization of this approach. Vertebrates have evolved three paralogs, termed LUC7L, LUC7L2 and LUC7L3, of the essential yeast U1 snRNA-associated splicing factor Luc7p. Only LUC7L2 is mutated or deleted in myeloid neoplasms. Notably, these paralogs share two conserved zinc finger motifs with Luc7p while adding divergent arginine-serine rich domains. The highly conserved second zinc finger domain of Luc7p stabilizes the binding of weak 5′ splice sites (5′SS) by directly contacting U1 snRNA at the 5′SS-U1 snRNA duplex. We investigated the mechanistic and regulatory functions of the LUC7-like family of splicing factors using a multi-omic approach to characterize protein-protein interactions, RNA binding profiles, regulated gene expression and alternative splicing events. Protein interaction data showed that all three proteins bound similar core but distinct regulatory splicing factors, likely mediated through their divergent arginine-serine rich domains. RNA binding studies revealed that LUC7L2 and LUC7L3 interacted with SR proteins and crosslinked to weak 5′SSs and to the 5′ end of U1 snRNA, establishing an evolutionarily conserved role in 5′SS selection. In contrast, LUC7L bound within introns and interacted primarily with hnRNP proteins. Knockdown of each factor revealed mostly unique sets of significantly dysregulated alternative splicing events dependent on their binding locations, which are largely non-overlapping. Knockdown of LUC7L2 alone significantly upregulated the expression of multiple spliceosomal factors and downregulated glycolysis genes, possibly contributing to disease pathogenesis. Overall, human LUC7-like proteins are components of the U1 snRNP that regulate unique alternative splicing profiles. Furthermore, LUC7L2 and LUC7L3 share an evolutionarily conserved role in 5′SS selection distinct from LUC7L.
Richard Padgett, Ph.D. (Advisor)
Donal Luse, Ph.D. (Committee Chair)
Angela Ting, Ph.D. (Committee Member)
Yogen Saunthararajah, M.D. (Committee Member)
George Stark, Ph.D. (Committee Member)
242 p.
Biochemistry; Bioinformatics; Biology; Biomedical Research; Cellular Biology; Genetics; Molecular Biology
LUC7L; LUC7L2; LUC7L3; DDX41; Myeloid Neoplasms; MDS; AML; alternative splicing; 5′ splice site; U1 snRNP; pre-mRNA splicing; SR protein

Recommended Citations

Citations

  • Daniels, N. J. (2022). Functional Analyses of Human DDX41 and LUC7-like Proteins Involved in Splicing Regulation and Myeloid Neoplasms [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1648820426769147

    APA Style (7th edition)

  • Daniels, Noah. Functional Analyses of Human DDX41 and LUC7-like Proteins Involved in Splicing Regulation and Myeloid Neoplasms. 2022. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1648820426769147.

    MLA Style (8th edition)

  • Daniels, Noah. "Functional Analyses of Human DDX41 and LUC7-like Proteins Involved in Splicing Regulation and Myeloid Neoplasms." Doctoral dissertation, Case Western Reserve University, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=case1648820426769147

    Chicago Manual of Style (17th edition)

case1648820426769147
155
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.
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