Mutations in the Saccharomyces cerevisiae LSM1 Gene That Affect mRNA Decapping and 3′ End Protection

Abstract

The decapping of eukaryotic mRNA s is a key step in their degradation. The heteroheptameric L sm1p-7p complex is a general activator of decapping and also functions in protecting the 3Ј ends of deadenylated mRNA s from a 3Ј-trimming reaction. L sm1p is the unique member of the L sm1p-7p complex, distinguishing that complex from the functionally different L sm2p-8p complex. To understand the function of L sm1p, we constructed a series of deletion and point mutations of the LSM1 gene and examined their effects on phenotype. These studies revealed the following: (i) Mutations affecting the predicted RNAbinding and inter-subunit interaction residues of L sm1p led to impairment of mRNA decay, suggesting that the integrity of the L sm1p-7p complex and the ability of the L sm1p-7p complex to interact with mRNA are important for mRNA decay function; (ii) mutations affecting the predicted RNA contact residues did not affect the localization of the L sm1p-7p complex to the P-bodies; (iii) mRNA 3Ј-end protection could be indicative of the binding of the L sm1p-7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3Ј end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of L sm1p is also important for mRNA decay function.

Lsm1-7-Pat1 complex: A link between 3’ and 5’-ends in mRNA decay?

“…Consistently, mRNAs undergo trimming at their 3'-ends in yeast lsm1 mutants and genetic analyses suggest that the temperature sensitivity of these mutants could be related to the enhanced exosome mediated 3' to 5' decay of the trimmed mRNAs. 36,53 These observations support the idea that apart from actively promoting the 5' to 3' involves poly(A) shortening to an intermediate tail length of ~110 A-residues and the mRNAs with A 110 tails are further deadenylated to oligoadenylated mRNAs in the second phase mediated by the Ccr4 deadenylase. 9 Interestingly a fraction of mRNAs is decapped after the first phase of deadenylation when the A-tails are ~110 nucleotides long while another fraction is decapped after the second phase of deadenylation.…”

“…39 Yet, these lsm1 mutants exhibit a clear mRNA decay defect in vivo. 36 These observations suggested that the oligo(A) tail mediated stimulation of Lsm1-7-Pat1 complex binding to mRNA is important for normal rates of mRNA decapping in vivo. Consistent with this, unadenylated mRNAs artificially generated in vivo via ribozyme cleavage are not efficiently targeted for Lsm1-mediated 5' to 3' decay 39 but readily undergo exosome mediated 3' to 5' decay.…”

“…These studies involved two lsm1 alleles that carry lesions in the predicted RNA binding residues of yeast Lsm1. 36 In vitro studies revealed that the mutant complexes purified from these lsm1 mutants fail to show higher affinity towards oligoadenylated RNAs over polyadenylated or unadenylated RNAs. 39 However, these mutant complexes are normal with regard to the complex integrity and their overall RNA binding ability is only moderately affected compared to the wild type complex.…”

“…[30][31][32][33][34] Loss of function mutations affecting any of these subunits result in stabilization of mRNAs in vivo by impairing mRNA decay via the 5' to 3' pathway without affecting deadenylation. 27,31,32,35,36 Oligoadenylated capped mRNAs accumulate in these mutants indicating that this complex promotes decapping following deadenylation in the 5' to 3' pathway and is therefore required for the normal rates of mRNA decapping in vivo. 31,32,35,36 In fact, immunoprecipitation analyses revealed that this complex preferentially associates with oligoadenylated mRNPs (but not polyadenylated mRNPs) that are also bound to the decapping enzyme (but not bound to key translation factors like the cap-binding complex).…”

“…27,31,32,35,36 Oligoadenylated capped mRNAs accumulate in these mutants indicating that this complex promotes decapping following deadenylation in the 5' to 3' pathway and is therefore required for the normal rates of mRNA decapping in vivo. 31,32,35,36 In fact, immunoprecipitation analyses revealed that this complex preferentially associates with oligoadenylated mRNPs (but not polyadenylated mRNPs) that are also bound to the decapping enzyme (but not bound to key translation factors like the cap-binding complex). 32,37 These observations further support the idea that the Lsm1-7-Pat1 complex targets the mRNA for decay in vivo after deadenylation.…”