Expression and analysis of the SAM-dependent RNA methyltransferase Rsm22 from Saccharomyces cerevisiae

Area of Science:

• Mitochondrial gene expression
• Protein structure and function
• RNA modification

Background:

• Saccharomyces cerevisiae Rsm22 (Sc-Rsm22) is homologous to Tb-Rsm22 and Mm-METTL17, all linked to mitochondrial gene expression.
• Sc-Rsm22 is essential for mitochondrial respiration, but its precise function and structure were unknown.
• Rsm22-family proteins' physiological roles and structural details remain largely uncharacterized.

Purpose of the Study:

• To elucidate the structure and function of Saccharomyces cerevisiae Rsm22 (Sc-Rsm22).
• To investigate the role of Sc-Rsm22 in mitochondrial gene expression and protein synthesis.
• To characterize the unique structural features of Sc-Rsm22 compared to its homologs.

Main Methods:

• Expression and purification of Sc-Rsm22 in monomeric and dimeric forms.
• Circular-dichroism analyses to confirm protein folding.
• Small-angle X-ray scattering (SAXS) to determine low-resolution structures.
• Bioinformatic analysis of protein domains and sequence similarity.
• In vitro functional assays to assess tRNA methylation activity.

Main Results:

• The monomeric Sc-Rsm22 structure revealed an elongated, three-domain arrangement distinct from Tb-Rsm22.
• A unique structural motif, including a Rossmann-like methyltransferase fold and a zinc-finger-like structure, was identified in the core domain.
• The N-terminal domain is predicted to be α-helical, sharing no sequence similarity with other family members.
• Functional studies confirmed that monomeric Sc-Rsm22 methylates mitochondrial tRNAs in vitro.

Conclusions:

• Sc-Rsm22 is a novel RNA methyltransferase essential for mitochondrial protein synthesis.
• The unique structure of Sc-Rsm22, particularly its N-terminal domain and zinc-finger-like motif, distinguishes it within the Rsm22 family.
• These findings provide critical insights into the mechanisms of mitochondrial gene expression regulation.