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 #
5SC1GM083754-02
Application #
7752587
Study Section
Special Emphasis Panel (ZGM1-MBRS-7 (GC))
Program Officer
Bender, Michael T
Project Start
2009-01-01
Project End
2012-12-31
Budget Start
2010-01-01
Budget End
2010-12-31
Support Year
2
Fiscal Year
2010
Total Cost
$269,500
Indirect Cost
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
Patel, Joy; Lama, Lodoe; Hoffmann, Niklas A et al. (2017) RNA polymerase III initiation on coligo DNA templates containing loops of variable sequence, size and nucleotide chemistry. Gene 612:49-54
Na, Mihwa; Valente, Susana T; Ryan, Kevin (2017) Optimizing In Vitro Pre-mRNA 3' Cleavage Efficiency: Reconstitution from Anion-Exchange Separated HeLa Cleavage Factors and from Adherent HeLa Cell Nuclear Extract. Methods Mol Biol 1507:179-198
Lama, Lodoe; Ryan, Kevin (2016) Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I. RNA 22:155-61
Khleborodova, Asya; Pan, Xiaozhou; Nagre, Nagaraja N et al. (2016) An investigation into the role of ATP in the mammalian pre-mRNA 3' cleavage reaction. Biochimie 125:213-22
Li, Yadi; Peterlin, Zita; Ho, Jianghai et al. (2014) Aldehyde recognition and discrimination by mammalian odorant receptors via functional group-specific hydration chemistry. ACS Chem Biol 9:2563-71
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
Lama, Lodoe; Seidl, Christine I; Ryan, Kevin (2014) New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III. Transcription 5:e27913
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 :
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

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