Genetic & Genomic Analysis of the mRNA 3' End Processing Subunit Pcf11 Suggests an Expanded Role in Gene Expression.
of mature mRNA is a multistep process requiring many proteins that is essential for
proper cellular function. Defects in mRNA maturation lead to radical changes in
development, growth and viability of the cell. The essential mRNA 3' end processing
subunit, Pcf11, is required for the cleavage and polyadenylation of nascent mRNAs and
for proper termination of RNA polymerase... read moreII transcription. Pcf11 also plays a role in
alternative polyadenylation. Previous work has identified and described the function of
several domains in the Pcf11 protein, but the crystal structure has not been solved and
there remain large stretches of Pcf11 that are uncharacterized. Pcf11 is part of the CF
1A factor involved in cleavage and polyadenylation. As part of CF 1A, Pcf11 makes
contacts with each of the other CF 1A protein subunits as well as several of the protein
subunits that make up the Cleavage and Polyadenylation Factor (CPF), but the importance
of these cross-factor interactions is not known. Pcf11 and other mRNA 3' end processing
subunits have been primarily studied in the context of RNA polymerase II transcription
and mRNA processing but there are indications that mRNA processing subunits participate
in other aspects of RNA maturation such as tRNA splicing. However, the function of mRNA
3' end processing factors in tRNA biology is entirely unknown. In studies described in
this thesis, we extensively analyzed a novel Pcf11 allele lacking the conserved amino
acids 142-225 and observed a growth defect on caffeine and transcription termination
defects at snoRNAs. We used Next generation direct RNA sequencing experiment to
determine the effect of loss of Pcf11 amino acids 142-225 genome wide and found that,
under standard growth conditions, the Pcf11 deletion mutant had RNAs from multiple
pathways that were upregulated compared to wild-type. Our data demonstrated that in
wild-type cells, half of the genes we examined used a cluster of putative poly(A) sites
proximal to the promoter of the gene. These promoter-proximal sites were suppressed in
cells lacking amino acids 142-225 of Pcf11. These results suggest a novel role for Pcf11
in the negative regulation of some genes by promoting the usage of a promoter-proximal
poly(A) sites. Finally, we demonstrated that mutant alleles of Pcf11 accumulate
unspliced tRNAs and show that amino acids 288-400 of Pcf11 are required for interaction
with the CPF subunit, Pta1. All together, the findings presented here demonstrate a
novel role for amino acids 142-225 of Pcf11 in the regulation of a number of genes from
diverse cellular pathways. We have also expanded Pcf11's role in RNA maturation by
implicating it in tRNA biology. Finally, we have done preliminary experiments to dissect
the domains important for interaction between Pcf11 and Pta1, and these findings will
guide future experiments to probe the importance of cross-factor interactions in RNA
Thesis (Ph.D.)--Tufts University, 2014.
Submitted to the Dept. of Genetics.
Advisor: Claire Moore.
Committee: Erik Selsing, Gordon Huggins, and Grace Gill.
Keyword: Genetics.read less