Leveraging rRNA pseudouridylation defects to identify operational programmed -1 ribosomal frameshift signals in yeast, mice and human cells.
EMSL Project ID
46896
Abstract
Programmed -1 ribosomal frameshifting (-1 PRF) is a molecular mechanism in which cis-acting signals direct a fraction of elongating ribosomes to slip by 1 nucleotide in the 5-prime direction along an mRNA. While first discovered in viruses, it is now clear that many cellular mRNAs also use -1 PRF to control gene expression. We have demonstrated an inverse correlation between the rate of -1 PRF an mRNA stability in cellular mRNAs. Specifically, increases in -1 PRF lead to decreases in mRNA stability, and consequently, decreased expression of proteins encoded by -1 PRF signal containing mRNAs. This relationship between -1 PRF efficiency and cellular gene expression is conserved from yeast to humans. Computational methods predict that ~10% of all cellular mRNAs may be regulated by -1 PRF, suggesting that -1 PRF may control the expression of a significant fraction of the genomes. While we have amassed a web-based community resource of putative cellular -1 PRF signal containing cellular genes (PRFdB), an -omics scale approach is needed to identify those that are actually employed. Here, deep sequencing methods will be applied to mutants in yeast, mouse and human cells that are known to promote increased or decreased rates of -1 PRF. Analysis of the data will identify those mRNAs that are less abundant when -1 PRF is stimulated and more abundant when it is inhibited. The experimental data and conclusions will be added to the current PRFdb, aiding biologists in the selection of particular genes for further investigation of control by embedded -1 PRF signal. This work will also open a new research route via broad comparative analysis as to why some putative -1 PRF signals are used while others are not. In addition, we have recently shown that mutants in the gene encoding the ribosomal RNA (rRNA) pseudouridine synthetase (called CBF5 in yeast, and DKC1 in mammals) promote globally increased rates of -1 PRF, resulting in decreased abundance of -1 PRF signal containing cellular mRNAs. Importantly, the -1 PRF stimulatory properties of CBF5/DKC1 mutants is conserved from yeast to humans. DKC1 is so named because mutations in this gene are the cause of the inherited disorder X-linked dyskeratosis congenita (X-DC). We hypothesize globally increased -1 PRF may be, in part, responsible for the pathologies associated with X-DC. Whole transcriptome data collected from mutant and wild-type DKC1 cells will identify those mRNAs whose expression is affected in diseased cells, laying a new groundwork for development of therapeutic strategies for X-DC.
Project Details
Project type
Exploratory Research
Start Date
2012-03-28
End Date
2013-04-07
Status
Closed
Released Data Link
Team
Principal Investigator