Biomedical subjects

S Tharun

Publications and source records attributed to S Tharun.

4 recordsLinked to original sources

PubMed2001

Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs.

The major pathway of eukaryotic mRNA decay involves deadenylation-dependent decapping followed by 5' to 3' exonucleolytic degradation. By examining interactions among mRNA decay factors, the mRNA, and key translation factors, we have identified a critical transition in mRNP organization that leads to decapping and degradation of yeast mRNAs. This transition occurs after deadenylation and includes loss of Pab1p, eIF4E, and eIF4G from the mRNA and association of the decapping activator complex, Lsm1p-7p, which enhances the coimmunoprecipitation of a decapping enzyme complex (Dcp1p and Dcp2p) with the mRNA. These results define an important rearrangement in mRNP organization and suggest that deadenylation promotes mRNA decapping by both the loss of Pab1p and the recruitment of the Lsm1p-7p complex.

DNA-Binding Proteins↗

PubMed2000

Yeast Sm-like proteins function in mRNA decapping and decay.

One of the main mechanisms of messenger RNA degradation in eukaryotes occurs by deadenylation-dependent decapping which leads to 5'-to-3' decay. A family of Sm-like (Lsm) proteins has been identified, members of which contain the 'Sm' sequence motif, form a complex with U6 small nuclear RNA and are required for pre-mRNA splicing. Here we show that mutations in seven yeast Lsm proteins (Lsm1-Lsm7) also lead to inhibition of mRNA decapping. In addition, the Lsm1-Lsm7 proteins co-immunoprecipitate with the mRNA decapping enzyme (Dcp1), a decapping activator (Pat1/Mrt1) and with mRNA. This indicates that the Lsm proteins may promote decapping by interactions with the mRNA and the decapping machinery. In addition, the Lsm complex that functions in mRNA decay appears to be distinct from the U6-associated Lsm complex, indicating that Lsm proteins form specific complexes that affect different aspects of mRNA metabolism.

Autoantigens↗

PubMed1999

Analysis of mutations in the yeast mRNA decapping enzyme.

A major mechanism of mRNA decay in yeast is initiated by deadenylation, followed by mRNA decapping, which exposes the transcript to 5' to 3' exonucleolytic degradation. The decapping enzyme that removes the 5' cap structure is encoded by the DCP1 gene. To understand the function of the decapping enzyme, we used alanine scanning mutagenesis to create 31 mutant versions of the enzyme, and we examined the effects of the mutations both in vivo and in vitro. Two types of mutations that affected mRNA decapping in vivo were identified, including a temperature-sensitive allele. First, two mutants produced decapping enzymes that were defective for decapping in vitro, suggesting that these mutated residues are required for enzymatic activity. In contrast, several mutants that moderately affected mRNA decapping in vivo yielded decapping enzymes that had at least the same specific activity as the wild-type enzyme in vitro. Combination of alleles within this group yielded decapping enzymes that showed a strong loss of function in vivo, but that still produced fully active enzymes in vitro. This suggested that interactions of the decapping enzyme with other factors may be required for efficient decapping in vivo, and that these particular mutations may be disrupting such interactions. Interestingly, partial loss of decapping activity in vivo led to a defect in normal deadenylation-dependent decapping, but it did not affect the rapid deadenylation-independent decapping triggered by early nonsense codons. This observation suggested that these two types of mRNA decapping differ in their requirements for the decapping enzyme.

Alleles↗

PubMed1995

Specific endonucleolytic cleavages of mouse albumin mRNA and their modulation during liver development.

We have detected specific endonucleolytic cleavages of mouse albumin mRNA by S1 nuclease protection analysis of total RNA from fetal mouse liver using a cDNA probe spanning the middle, coding region of albumin mRNA. With the use of probe labeled at its 5' end, three prominent cleavages were detected which were confirmed and their endonucleolytic nature was established by further analysis using 3' end-labeled probe. The latter probe also revealed one more cleavage which was not detected with the 5' end-labeled probe. These cleavages mapped to positions on the mRNA which included a unique sequence motif CCAN1-3CUGN0-1UGAU. Degradation intermediates corresponding to these cleavages were consistently observed, specifically in fetal liver but not in normal or regenerating adult liver and appeared to have originated in vivo. Their levels decreased progressively from 18th day of gestation and became undetectable by 20 days after birth. No detectable changes in the levels of any of the prominent degradation products of alpha-fetoprotein (a homologue of albumin) mRNA could be observed during this period of development. Since accumulation of degradation intermediates is known to correlate with higher rate of mRNA turnover, our observations raise the possibility that the stability of albumin mRNA may be lower in fetal than in adult mouse liver.

Age Factors↗