Journal: RNA Biology
Article Title: Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence
Figure Lengend Snippet: Sgd1 crosslinks to helix H12 of the 18S rRNA sequence . (A) Wild type yeast (WT) or a yeast strain expressing C-terminally His-TEV protease cleavage site-Protein A (HTP) tagged Sgd1 from its genomic locus were crosslinked in culturo using irradiation at 254 nm. The tagged protein and crosslinked RNAs were retrieved under native conditions on IgG sepharose and subjected to a partial RNase digest. Complexes were then enriched under denaturing conditions on NiNTA, and co-purified RNA fragments were [ 32 P] labelled and ligated to sequencing adaptors. Protein-RNA complexes were separated by denaturing PAGE, transferred to a nitrocellulose membrane and radiolabelled RNAs were detected by autoradiography. A non-specific signal is indicated by an asterisk. (B) The region of the membrane containing crosslinked Sgd1-HTP complexes, and a corresponding region of the lane containing the WT sample, were excised and RNAs were release by protease treatment. Purified RNAs were converted to a cDNA library that was subjected to deep sequencing. The obtained sequencing reads were mapped to the S. cerevisiae genome and the relative proportions of reads mapped to gene features encoding different classes of RNA was determined. Abbreviations – ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), small nuclear RNA (snRNA). (C) After normalization, the number of reads mapping to each nucleotide of RDN37 , which encodes the 35S pre-rRNA transcript, was determined for the WT and Sgd1-HTP samples and is shown above a schematic view of the pre-rRNA transcript. The number of mutations, induced by the presence of crosslinked nucleotides, mapping to each nucleotide is also shown. (D) The number of sequencing reads in the Sgd1-HTP CRAC dataset mapping to each nucleotide of the 18S rRNA sequence is shown on the secondary structure of the mature rRNA using a colour scale in which the maximum number of reads (100%) is highlighted in red and lesser numbers of reads ( > 20%) are shown in yellow. A magnified view of the region of the 18S rRNA to which Sgd1 binds is presented. (E) The number of sequencing reads mapping to each nucleotide of the 18S rRNA sequence was mapped onto the tertiary structure of the SSU processome (PBD: 5WLC) using a colour scale as in (D). Pre-rRNA sequences are shown in cartoon mode, and ribosomal proteins and ribosome assembly factors are shown in surface view in pale cyan. The densities corresponding to specific ribosomal proteins and ribosome assembly factors that are present in close proximity to the crosslinking site of Sgd1 are highlighted.
Article Snippet: For recombinant expression of Sgd1 in E. coli , a codon-optimized version of the Sgd1 coding sequence was synthesized by MWG Eurofins and sub-cloned into a pQE80-derived plasmid for the expression of an N-terminally MBP-TEV protease cleavage site, C-terminally His tagged protein.
Techniques: Sequencing, Expressing, Irradiation, Purification, Polyacrylamide Gel Electrophoresis, Autoradiography, cDNA Library Assay