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

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

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

Effect of the C-terminal extension mutations of the <i>lsm1-39</i>, <i>lsm1-40</i> and <i>lsm1-41</i> alleles on mRNA decay.

<p>RNA isolated from <i>lsm1Δ</i> cells expressing wild type or various mutant alleles of <i>LSM1</i> (left panel) or from <i>lsm1-27</i> cells expressing wild type or various mutant versions of the C-terminal extension peptide of Lsm1 from multi copy 2μ vectors (right panel) were subjected to Northern analysis to reveal the <i>MFA2pG</i> mRNA and the poly(G) fragment. Poly(G) fragment levels were approximated and presented as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158876#pone.0158876.g002" target="_blank">Fig 2</a>.</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