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.
|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|
|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 :|
|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|
|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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