Nearly all eukaryotic pre-messenger RNAs must undergo cleavage and polyadenylation in their 3'-untranslated region (UTR) before being exported to the cytoplasm for translation into protein. The correct execution of these coupled and cotranscriptional processing events is critical for proper gene expression and normal cellular life. This topic has relevance to human health: certain heritable diseases cause illness through improper cleavage and polyadenylation. For example, the 1- and 2-thalassemias are caused by mutations in the processing signals of the 3'UTR of the globin genes. Approximately half of all human genes can be polyadenylated at different locations, a fact that can have regulatory consequences for an mRNA and its gene product, but little is known about the biochemical mechanisms by which such alternative polyadenylation takes place. We have previously found that a chromatography fraction containing both mammalian Cleavage Factors I and II (CFIm/CFIIm) loses activity when subjected to dephosphorylating enzymes in vitro. We propose that phosphorylation among these protein factors may play a role in their function and/or regulation. Our long-term goal is to understand how post-translational phosphorylation among the pre-mRNA 3'cleavage factors contributes to both the constitutive and regulated versions of this essential biochemical reaction.
Our specific aims for this work are: 1. To identify the human cleavage factor whose activity is determined by reversible phosphorylation? 2. To make and test recombinant CFIm heterodimers for phosphorylation-dependent in vitro reconstituted 3'cleavage, and cleavage rescue, activity. 3. To perform a mutational analysis of 3'cleavage activity dependence on CFIm phosphorylation. 4. To study pre-cleavage complex formation as a function of protein phosphorylation. Project Narrative This project will study the 3'cleavage and polyadenylation of messenger RNA. This topic has relevance to human health: certain heritable diseases cause illness through improper cleavage and polyadenylation. For example, the 1- and 2-thalassemias are caused by mutations in the processing signals of the 3'UTR of the globin genes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Enhancement Award (SC1)
Project #
5SC1GM083754-04
Application #
8209004
Study Section
Special Emphasis Panel (ZGM1-MBRS-7 (GC))
Program Officer
Bender, Michael T
Project Start
2009-01-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2013-12-31
Support Year
4
Fiscal Year
2012
Total Cost
$266,805
Indirect Cost
$93,555
Name
City College of New York
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
603503991
City
New York
State
NY
Country
United States
Zip Code
10031
Jablonski, Joseph; Clementz, Mark; Ryan, Kevin et al. (2014) Analysis of RNA processing reactions using cell free systems: 3' end cleavage of pre-mRNA substrates in vitro. J Vis Exp :
Liu, Min Ting; Nagre, Nagaraja N; Ryan, Kevin (2014) Structurally diverse low molecular weight activators of the mammalian pre-mRNA 3' cleavage reaction. Bioorg Med Chem 22:834-41
Seidl, Christine I; Lama, Lodoe; Ryan, Kevin (2013) Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells. Nucleic Acids Res 41:2552-64
Seidl, Christine I; Ryan, Kevin (2011) Circular single-stranded synthetic DNA delivery vectors for microRNA. PLoS One 6:e16925
Kurland, Michael D; Newcomer, Michael B; Peterlin, Zita et al. (2010) Discrimination of saturated aldehydes by the rat I7 olfactory receptor. Biochemistry 49:6302-4
Ryan, Kevin; Khleborodova, Asya; Pan, Jingyi et al. (2009) Small molecule activators of pre-mRNA 3'cleavage. RNA 15:483-92