Uniformed Services University of the Health Sciences
Department of Biochemistry and Molecular Biology
4301 Jones Bridge Road, C1094
Bethesda, Maryland 20814-4799
Fax: (301) 295-3512
Lab: (301) 295-3576 Email: firstname.lastname@example.org
Role of Sm-like proteins in mRNA decay.
The long term goal of our lab is to understand the mechanism of mRNA decay in eukaryotes using the budding yeast S. cerevisiae as the model system. mRNA decay is a critical determinant of gene expression and deregulation of mRNA decay is known to be the cause of several human diseases including cancer.
The mRNA decay pathways and decay factors are well conserved in all eukaryotes from yeast to humans. Two major pathways of mRNA decay exist in eukaryotes. Both pathways are initiated by poly(A) shortening of the mRNA. In the 5' to 3' pathway, this is followed by decapping which then permits the 5' to 3' exonucleolytic degradation of the message body. In the 3' to 5' decay pathway, deadenylated mRNAs are degraded in a 3' to 5' exonucleolytic manner.
In the 5' to 3' pathway, decapping is a crucial precisely controlled step affected by numerous factors. Oligoadenylated mRNAs but not polyadenylated mRNAs are selectively targeted for decapping in the 5' to 3' decay pathway resulting in the deadenylation dependence of decapping in this pathway. While translation initiation factors are antagonistic to the decapping enzyme, several other factors enhance the decapping enzyme function in vivo.
The Lsm1p-7p-Pat1p complex (made of seven Sm-like proteins, Lsm1p through Lsm7p and Pat1p) is a key activator of decapping needed for normal rates of decapping in vivo. It is conserved in all eukaryotes and interacts with several decay factors and with the mRNA in vivo. We showed earlier that the Lsm1p-7p-Pat1p complex selectively associates with mRNPs targeted for decay but not with translating mRNPs. Studies in our lab are focused towards understanding the mechanism by which this complex functions.
We purified the native Lsm1-7-Pat1 complex from yeast and showed that it has an intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs (as revealed by its several fold higher affinity for the former than the latter) in vitro suggesting that this complex plays a key role in the selective targeting of oligoadenylated mRNAs for decapping in vivo in the 5' to 3' pathway. Such ability of this complex is indeed crucial for the in vivo function of this complex in mRNA decay because, point mutations in LSM1 gene that abolish such ability (without affecting the other RNA binding characteristics of the complex or the complex integrity) result in strong mRNA decay defects in vivo. By studying multiple lsm1 mutants that are impaired in mRNA decay in vivo and determining the stage at which each of them is blocked using a variety of genetic and biochemical analyses, we also showed that decapping activation by the Lsm1-7-Pat1 complex in vivo requires both the binding of that complex to the mRNA and facilitation of one or more (unknown) post-binding events.
The Lsm1 through Lsm7 subunits of the Lsm1-7-Pat1 complex belong to the family of Sm-like proteins characterized by the presence of the Sm-domain. Lsm1 is a key subunit that critically determines the function of the Lsm1-7-Pat1 complex. As mentioned above our mutational analyses have revealed that the RNA binding residues in the Sm-domain of Lsm1 are crucial for the in vivo functions and unique in vitro RNA binding properties of the Lsm1-7-Pat1 complex. However, unlike many other Sm-like proteins, yeast Lsm1 has a long C-terminal domain (CTD) following its Sm-domain and this feature is conserved in human Lsm1 also. Interestingly our studies showed that the CTD of Lsm1 is also necessary (in addition to the Sm-domain of Lsm1) for the in vivo functions and the RNA binding activity of the Lsm1-7-Pat1 complex. Additional studies also showed that the CTD of Lsm1 could even act in trans to support the function of the Lsm1-7-Pat1 complex in vivo suggesting that it folds as a separate domain in the Lsm1 subunit. Thus Lsm1 is a unique Sm-like protein whose functions are determined not just by its Sm-domain but also residues outside the Sm-domain.
Recently we have focused our studies on the Pat1 subunit of the Lsm1-7-Pat1 complex. Our studies support the idea that RNA binding and recognition of the oligo(A) tail by this complex involve composite binding surfaces made of residues from both Pat1 and Lsm1. Our studies also revealed that contrary to what was believed, the C-terminal domain of Pat1 is not sufficient for association with the Lsm1-7 ring and that the middle domain is also needed.
Chowdhury, A., Kalurupalle, S., and Tharun, S*. 2014. Pat1 contributes to the RNA binding activity of the Lsm1-7-Pat1 complex. RNA, 20:1465-1475.
Chowdhury, A., Raju, KK., Kalurupalle, S., and Tharun, S. 2012. Both Sm-domain and C-terminal extension of Lsm1 are important for the RNA-binding activity of the Lsm1-7-Pat1 complex. RNA, 18:936-944.
Chowdhury, A., and Tharun, S. 2009. Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7-Pat1 complex. RNA, 15:1837-1848.
Tharun, S. 2009. Lsm1-7-Pat1 complex: A link between 3' and 5'-ends in mRNA decay? RNA Biology, 6(3):228-232.
Tharun, S. 2009. Roles of eukaryotic Lsm proteins in the regulation of mRNA function. International Review of Cell & Molecular Biology, 272:149-89.
Tharun, S. 2008. Purification and analysis of the decapping activator Lsm1p-7p-Pat1p complex from yeast. Methods in Enzymology, 448:41-55.
Chowdhury, A., and Tharun, S. 2008. lsm1 mutations impairing the ability of the Lsm1p-7p-Pat1p complex to preferentially bind to oligoadenylated RNA affect mRNA decay in vivo. RNA, 14:2149-2158.
Chowdhury, A., Mukhopadhyay, J., Tharun, S. 2007. The decapping activator Lsm1p-7p-Pat1p complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs. RNA, 13:998-1016.
Tharun S*., Muhlrad D., Chowdhury A, Parker R. 2005. Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3' end protection. Genetics, 170:33-46 (*Corresponding author).
Tharun, S*., and Parker R. 2001. Targeting an mRNA for decapping: Displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs. Molecular Cell, 8:1075-1083 (*Corresponding author).
Tharun, S., He, W., Meyes, A., Lennertz, P., Beggs, J., and Parker, R. 2000. Yeast Sm-like proteins function in mRNA decapping and decay. Nature, 404:515-518.
Tharun, S., and Parker, R. 1999. Analysis of mutations in the yeast mRNA decapping enzyme. Genetics, 151:1273-1285.