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' decay1, 2. 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 splicing3-9. 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

lsm1 mutations impairing the ability of the Lsm1p-7p-Pat1p complex to preferentially bind to oligoadenylated RNA affect mRNA decay in vivo

The poly(A) tail is a crucial determinant in the control of both mRNA translation and decay. Poly(A) tail length dictates the triggering of the degradation of the message body in the major 5′ to 3′ and 3′ to 5′ mRNA decay pathways of eukaryotes. In the 5′ to 3′ pathway oligoadenylated but not polyadenylated mRNAs are selectively decapped in vivo, allowing their subsequent degradation by 5′ to 3′ exonucleolysis. The conserved Lsm1p-7p-Pat1p complex is required for normal rates of decapping in vivo, and the purified complex exhibits strong binding preference for oligoadenylated RNAs over polyadenylated or unadenylated RNAs in vitro. In the present study, we show that two lsm1 mutants produce mutant complexes that fail to exhibit such higher affinity for oligoadenylated RNA in vitro. Interestingly, these mutant complexes are normal with regard to their integrity and retain the characteristic RNA binding properties of the wild-type complex, namely, binding near the 3′-end of the RNA, having higher affinity for unadenylated RNAs that carry U-tracts near the 3′-end over those that do not and exhibiting similar affinities for unadenylated and polyadenylated RNAs. Yet, these lsm1 mutants exhibit a strong mRNA decay defect in vivo. These results underscore the importance of Lsm1p-7p-Pat1p complex–mRNA interaction for mRNA decay in vivo and imply that the oligo(A) tail mediated enhancement of such interaction is crucial in that process

Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7–Pat1 complex

Decapping is a critical step in the conserved 5′-to-3′ mRNA decay pathway of eukaryotes. The hetero-octameric Lsm1-7–Pat1 complex is required for normal rates of decapping in this pathway. This complex also protects the mRNA 3′-ends from trimming in vivo. To elucidate the mechanism of decapping, we analyzed multiple lsm1 mutants, lsm1-6, lsm1-8, lsm1-9, and lsm1-14, all of which are defective in decapping and 3′-end protection but unaffected in Lsm1-7–Pat1 complex integrity. The RNA binding ability of the mutant complex was found to be almost completely lost in the lsm1-8 mutant but only partially impaired in the other mutants. Importantly, overproduction of the Lsm1-9p- or Lsm1-14p-containing (but not Lsm1-8p-containing) mutant complexes in wild-type cells led to a dominant inhibition of mRNA decay. Further, the mRNA 3′-end protection defect of lsm1-9 and lsm1-14 cells, but not the lsm1-8 cells, could be partly suppressed by overproduction of the corresponding mutant complexes in those cells. These results suggest the following: (1) Decapping requires both binding of the Lsm1-7–Pat1 complex to the mRNA and facilitation of the post-binding events, while binding per se is sufficient for 3′-end protection. (2) A major block exists at the post-binding steps in the lsm1-9 and lsm1-14 mutants and at the binding step in the lsm1-8 mutant. Consistent with these ideas, the lsm1-9, 14 allele generated by combining the mutations of lsm1-9 and lsm1-14 alleles had almost fully lost the RNA binding activity of the complex and behaved like the lsm1-8 mutant

The decapping activator Lsm1p-7p–Pat1p complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs

Decapping is a critical step in mRNA decay. In the 5′-to-3′ mRNA decay pathway conserved in all eukaryotes, decay is initiated by poly(A) shortening, and oligoadenylated mRNAs (but not polyadenylated mRNAs) are selectively decapped allowing their subsequent degradation by 5′ to 3′ exonucleolysis. The highly conserved heptameric Lsm1p-7p complex (made up of the seven Sm-like proteins, Lsm1p–Lsm7p) and its interacting partner Pat1p activate decapping by an unknown mechanism and localize with other decapping factors to the P-bodies in the cytoplasm. The Lsm1p-7p–Pat1p complex also protects the 3′-ends of mRNAs in vivo from trimming, presumably by binding to the 3′-ends. In order to determine the intrinsic RNA-binding properties of this complex, we have purified it from yeast and carried out in vitro analyses. Our studies revealed that it directly binds RNA at/near the 3′-end. Importantly, it possesses the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs such that the former are bound with much higher affinity than the latter. These results indicate that the intrinsic RNA-binding characteristics of this complex form a critical determinant of its in vivo interactions and functions

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

The decapping of eukaryotic mRNAs is a key step in their degradation. The heteroheptameric Lsm1p–7p complex is a general activator of decapping and also functions in protecting the 3′ ends of deadenylated mRNAs from a 3′-trimming reaction. Lsm1p is the unique member of the Lsm1p–7p complex, distinguishing that complex from the functionally different Lsm2p–8p complex. To understand the function of Lsm1p, 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 RNA-binding and inter-subunit interaction residues of Lsm1p led to impairment of mRNA decay, suggesting that the integrity of the Lsm1p–7p complex and the ability of the Lsm1p–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 Lsm1p–7p complex to the P-bodies; (iii) mRNA 3′-end protection could be indicative of the binding of the Lsm1p–7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3′ end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of Lsm1p is also important for mRNA decay function

Mutagenic Analysis of the C-Terminal Extension of Lsm1 - Fig 1

<p><i>A</i>, Alignment of the C-terminal most 55 residues of <i>S</i>. <i>cerevisiae</i> and <i>P</i>. <i>stipitis</i> Lsm1 proteins. Numbers of the first and last residues are indicated on the left and right of the sequence. <i>B</i>, Three dimensional structure of Lsm1 subunit in the Lsm1-7 complex (PDB ID: 4C92; Sharif and Conti, 2013) is shown as ribbon diagram. Parts of the ribbon corresponding to some of the C-terminal extension residues targeted in the <i>lsm1</i> mutants are shown in red. For these residues the orientation of the side chains is also shown. Rest of the C-terminal extension is shown in green. The N-terminal extension and the Sm domain are shown in gray.</p

The C-terminal extension of Lsm1 is able to bind RNA by itself and the C-terminal most 8 residues are important for such binding.

<p><i>A</i>, Synthetic untagged wild type Lsm1 C-terminal extension peptide (10μM) or increasing concentrations (0.5μM, 2μM and 10μM) of synthetic 6xHis-tagged C-terminal extension peptides corresponding to <i>LSM1</i>, <i>lsm1-39</i>, <i>lsm1-40</i> or <i>lsm1-41</i> were incubated with radiolabeled <i>in vitro</i> transcript carrying the 3’-most 43 residues of the yeast <i>MFA2</i> mRNA and then subjected to pull down using the Ni-NTA matrix. After washing, the co-precipitated RNA was run alongside untreated RNA (10% of total amount used for the binding) and then visualized by denaturing PAGE and phosphorimaging. <i>B</i>, A plot of the percentage of RNA bound vs the concentration of the peptide used is shown. Plotted values are mean ± SD from three independent trials. <i>C</i>, <i>Top panel</i>, bovine serum albumin (BSA) or synthetic C-terminal segment peptides (2 nmols each) corresponding to <i>LSM1</i>, <i>lsm1-39</i> or <i>lsm1-41</i> were incubated with radiolabeled <i>in vitro</i> transcript carrying the 3’-most 43 residues of the yeast <i>PGK1</i> mRNA, UV crosslinked, treated with ribonuclease and then visualized by SDS-PAGE and phosphorimaging. <i>Bottom panel</i>, Similar UV crosslinking analysis carried out with synthetic wild type Lsm1 C-terminal extension peptide (untagged or 6xHis-tagged) or BSA is shown.</p

Suppression of the mRNA decay phenotype of the <i>lsm1-27</i> mutant (C-terminal truncation mutant of <i>LSM1</i>) in <i>trans</i> by the various mutant versions of the C-terminal extension peptide, upon over expression.

<p>RNA isolated from <i>lsm1-27</i> cells expressing wild type or various mutant versions of the C-terminal extension peptide of Lsm1 from multi copy <i>2μ</i> vectors were subjected to Northern analysis to reveal the <i>MFA2pG</i> mRNA and the poly(G) fragments. The fractional contribution of the poly(G) fragments to the total signal (total = full-length mRNA + trimmed and normal poly(G) fragments) was approximated for each sample via quantitation using the phosphorimager and normalized to the value obtained for <i>lsm1-27</i> cells expressing wild type C-terminal extension peptide. Samples with approximate poly(G) fragment levels that are ≥ 80%, 60% to 80% and <60% of the value for cells expressing wild type peptide are marked with +++, ++ and + below the corresponding lanes in the figure.</p

Residue changes in the various <i>lsm1</i> alleles generated in this study.

<p>Residue changes in the various <i>lsm1</i> alleles generated in this study.</p