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.

Abstract

<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