Splicing of pre-mRNAs provides a major source of transcript diversity for cell differentiation and development. The process of splicing requires a splicing machine known as the spliceosome that comprises ~100 proteins and five small nuclear (sn)RNAs. The early stage of spliceosome assembly on pre-mRNA splice sites is a key regulated step that often goes awry in human genetic diseases and cancers. Yet, exactly how the spliceosome selects and excises the correct splice sites from amidst thousands of competing pre-mRNA sequences remains poorly understood at the molecular level. The overall goal of this project is to understand how an essential complex comprising U2AF65, U2AF35, and SF1 proteins can accurately target 3'splice sites, leading to spliceosome assembly.
Specific aims of this proposal will test the following central hypotheses concerning the critical early stages of pre-mRNA splicing: Hypothesis #1: Our 'panoptic'understanding of U2AF65 recognition of 3'splice site RNAs - emerging from our new structures of intact U2AF65 as well as past work on core domains- can be used to understand pre-mRNA splice site mutations from specific human diseases. Hypothesis #2: We will expand our prior results and preliminary data to test the spliceosome subunit SF3b155 as a "molecular hub" coordinating phosphorylation-sensitive assemblies comprising U2AF65, the cancer-related factor paralogue CAPERa, and the p14 subunit, which in turn contacts the branch-site nucleophile of the spliceosome. Hypothesis #3: Our innovative SF1/U2AF65/U2AF35/RNA preparation positions us to locate the protein and RNA subunits in the complex, and to test the structural and functional effects of U2AF35 mutations that frequently cause myelodysplasia, hematological malignancies and lung cancer. Our overall approach entails a multi-front attack on all aims using a multidisciplinary strategy. Our core biophysical technologies entail X-ray crystallography, fluorescence anisotropy, isothermal titration calorimetry, and small-angle X-ray scattering with purified proteins. To meet challenges and propel the field in new directions, we will utilize a powerful combination of innovative methods including protein and RNA labeling, Forster resonance energy transfer, small-angle neutron scattering, and site-specific photo-crosslinking followed by LC-MS/MS. These tools are complemented by strong collaborations to study splicing factor functions in nuclear extracts and in living cells. Our research is grounded in the fundamental structure and function of 3'splice site recognition yet will broadly impact the field's understanding of aberran splicing, which is a dominant cause of blood disorders, neuromuscular diseases, leukemias and cancers.

Public Health Relevance

The U2AF65, U2AF35, and SF1 proteins are essential for splicing of human gene transcripts. We will determine three dimensional views and the molecular-level mechanism of action by these proteins. We specifically investigate errors in gene splicing that contribute to hematological malignancies and lung cancer. Our work offers a basis for understanding, and in the future developing treatments against, harmful splice variants of human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM070503-11A1
Application #
8696485
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter
Project Start
2004-07-01
Project End
2018-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
11
Fiscal Year
2014
Total Cost
$388,819
Indirect Cost
$135,517
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Loerch, Sarah; Maucuer, Alexandre; Manceau, Valérie et al. (2014) Cancer-relevant splicing factor CAPER? engages the essential splicing factor SF3b155 in a specific ternary complex. J Biol Chem 289:17325-37
Wang, Wenhua; Maucuer, Alexandre; Gupta, Ankit et al. (2013) Structure of phosphorylated SF1 bound to U2AFýýýýýý in an essential splicing factor complex. Structure 21:197-208
Bauer, William J; Heath, Jason; Jenkins, Jermaine L et al. (2012) Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1. J Mol Biol 415:727-40
Gupta, Ankit; Jenkins, Jermaine L; Kielkopf, Clara L (2011) RNA induces conformational changes in the SF1/U2AF65 splicing factor complex. J Mol Biol 405:1128-38
McLaughlin, Krystle J; Jenkins, Jermaine L; Kielkopf, Clara L (2011) Large favorable enthalpy changes drive specific RNA recognition by RNA recognition motif proteins. Biochemistry 50:1429-31
Gupta, Ankit; Kielkopf, Clara L (2011) Purification, crystallization and preliminary X-ray crystallographic analysis of a central domain of human splicing factor 1. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:486-90
Manceau, Valerie; Kielkopf, Clara L; Sobel, Andre et al. (2008) Different requirements of the kinase and UHM domains of KIS for its nuclear localization and binding to splicing factors. J Mol Biol 381:748-62
Jenkins, Jermaine L; Shen, Haihong; Green, Michael R et al. (2008) Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65. J Biol Chem 283:33641-9
Lin, Yuan; Kielkopf, Clara L (2008) X-ray structures of U2 snRNA-branchpoint duplexes containing conserved pseudouridines. Biochemistry 47:5503-14
Kumar, Amit O; Swenson, Matthew C; Benning, Matthew M et al. (2008) Structure of the central RNA recognition motif of human TIA-1 at 1.95A resolution. Biochem Biophys Res Commun 367:813-9

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