From yeast to humans, splicing is an essential step in the maturation of precursor messenger RNA (pre-mRNA). Anomalous pre-mRNA splicing can have lethal effects for the cell and has been linked to numerous human diseases such as cancer and neurodegenerative disorders. The spliceosome is a dynamic assembly of five small nucleolar RNAs (snRNA) and a large number of proteins that catalyzes splicing. Two snRNAs, U2 and U6, form the active site of the spliceosome. The structure of the U2/U6 complex has been the focus of much debate in recent years, because evidence has been presented for alternative conformations. We hypothesize that these conformations reflect different states of spliceosome activation, but specific structural dynamics information to support this hypothesis is still lacking. It is essential to study the structural dynamics of the U2/U6 complex to understand spliceosomal activation and catalysis, because this enzyme plays key roles in cell growth, differentiation and disease. We propose to use the powerful single molecule fluorescence technique to investigate these structural dynamics. We have previously demonstrated our approach to be particularly suited to elucidate the structural dynamics of RNA enzymes and reveal important information otherwise hidden in ensemble-averaged experiments.
We aim at (1) revealing the U2/U6 conformational dynamics by single molecule fluorescence, (2) elucidating the role of Mg2+ ions in these dynamics, (3) linking these dynamics to spliceosomal activation in vivo, (4) comparing the structural dynamics of the U2/U6 complex from humans and yeast and (5) elucidating the role of spliceosomal protein Prp24 in spliceosomal activation.

Public Health Relevance

From yeast to humans, splicing is an essential step in the maturation of messenger RNAs that are used to synthesize functional proteins. Anomalous splicing can have lethal effects for the cell and has been linked to numerous human diseases such as cancer and neurodegenerative disorders. The spliceosome is a large RNA-protein complex that catalyzes splicing. The structure of the catalytic center of the spliceosome has been a matter of debate in recent years because of its dynamic nature. It is essential to investigate the structural dynamics of the catalytic core of the spliceosome to understand its biological function, because this enzyme plays key roles in cell growth, differentiation and disease. We propose to use the powerful single molecule fluorescence technique to resolve these structural dynamics and characterize their mechanism in unprecedented detail.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085116-05
Application #
8266517
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Bender, Michael T
Project Start
2008-07-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2012
Total Cost
$265,001
Indirect Cost
$88,583
Name
Wayne State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
Karunatilaka, Krishanthi S; Rueda, David (2014) Post-transcriptional modifications modulate conformational dynamics in human U2-U6 snRNA complex. RNA 20:16-23
Mundigala, Hansini; Michaux, Jonathan B; Feig, Andrew L et al. (2014) HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway. Nucleic Acids Res 42:7281-9
Warnasooriya, Chandani; Rueda, David (2014) Single-molecule fluorescence-based studies on the dynamics, assembly and catalytic mechanism of the spliceosome. Biochem Soc Trans 42:1211-8
Brenlla, Alfonso; Markiewicz, Radoslaw P; Rueda, David et al. (2014) Nucleotide selection by the Y-family DNA polymerase Dpo4 involves template translocation and misalignment. Nucleic Acids Res 42:2555-63
Konig, Sebastian L B; Liyanage, Pramodha S; Sigel, Roland K O et al. (2013) Helicase-mediated changes in RNA structure at the single-molecule level. RNA Biol 10:133-48
Vrtis, Kyle B; Markiewicz, Radoslaw P; Romano, Louis J et al. (2013) Carcinogenic adducts induce distinct DNA polymerase binding orientations. Nucleic Acids Res 41:7843-53
Senavirathne, Gayan; Jaszczur, Malgorzata; Auerbach, Paul A et al. (2012) Single-stranded DNA scanning and deamination by APOBEC3G cytidine deaminase at single molecule resolution. J Biol Chem 287:15826-35
Salim, Nilshad; Lamichhane, Rajan; Zhao, Rui et al. (2012) Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex. Biophys J 102:1097-107
Cardo, Lucia; Karunatilaka, Krishanthi S; Rueda, David et al. (2012) Single molecule FRET characterization of large ribozyme folding. Methods Mol Biol 848:227-51
Wood, Sharla; Ferré-D'Amaré, Adrian R; Rueda, David (2012) Allosteric tertiary interactions preorganize the c-di-GMP riboswitch and accelerate ligand binding. ACS Chem Biol 7:920-7

Showing the most recent 10 out of 24 publications