Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence

Abstract

Assembly of eukaryotic ribosomal subunits is a complex and dynamic process involving the action of more than 200 transacting assembly factors. Although recent cryo-electron microscopy structures have provided information on architecture of several pre-ribosomal particles and the binding sites of many AFs, the RNA and protein interactions of many other AFs not captured in these snapshots still remain elusive. RNA helicases are key regulators of structural rearrangements within pre-ribosomal complexes and here we have analysed the eIF4A-like RNA helicase Fal1 and its putative cofactor Sgd1. Our data show that these proteins interact directly via the MIF4G domain of Sgd1 and that the MIF4G domain of Sgd1 stimulates the catalytic activity of Fal1 in vitro. The catalytic activity of Fal1, and the interaction between Fal1 and Sgd1, are required for efficient pre-rRNA processing at the A 0 , A 1 and A 2 sites. Furthermore, Sgd1 co-purifies the early small subunit biogenesis factors Lcp5 and Rok1, suggesting that the Fal1-Sgd1 complex likely functions within the SSU processome. In vivo crosslinking data reveal that Sgd1 binds to helix H12 of the 18S rRNA sequence and we further demonstrate that this interaction is formed by the C-terminal region of the protein, which is essential for its function in ribosome biogenesis.

The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly

“…RNA isolation and northern blotting. RNA was extracted from yeast cells, sucrose density gradient fractions, and pre-60S particles immobilized on IgG Sepharose using phenol-chloroform-based extraction 47,49,74 . For analysis of pre-rRNAs, 6 μg of total RNA or RNA isolated from pre-60S particles were separated on a denaturing (glyoxal) 1.2% ultrapure agarose gel and transferred by vacuum blotting onto a nylon membrane.…”

Regulation of DEAH-box RNA helicases by G-patch proteins

“…While many RNA helicases rely on dedicated cofactor proteins whose mode of interaction with the helicase is protein-specific (for example, Dbp8 and Esf2, DHX9 and EWS-FLI, Upf1 and Ufp2, and Rok1 and Rrp5 (Chakrabarti et al 2011;Erkizan et al 2015;Granneman et al 2006;Young et al 2013)), other cofactor proteins contain characteristic domains through which they interact with and regulate the activity of specific classes of RNA helicases. Several proteins containing a middle of eIF4G (MIF4G) domains regulate eIF4A-like RNA helicases (see for example, Alexandrov et al 2011;Davila Gallesio et al 2020;Mugler et al 2016;Schütz et al 2008). However, in this review, we focus on the G-patch proteins as another family of RNA helicase cofactors.…”

The RNA methyltransferase METTL8 installs m3C32 in mitochondrial tRNAsThr/Ser(UCN) to optimise tRNA structure and mitochondrial translation

“…For CRISPR/Cas9 constructs, guide sequences (Supplementary Table 6 ) were cloned into the PX459 plasmid pSpCas9(BB)-2A-Puro (Addgene #62988) using the BbsI restriction sites as previously described 75 . For recombinant expression of His 14 -MBP-METTL8 in E. coli , the CDS was cloned into a pQE80-derivative vector 76 and site-directed mutagenesis (Supplementary Table 6 ) was employed to introduce mutations to induce substitution of aspartates 230 and 309 with alanine. Plasmids containing tRNA sequences including a 3′ CCA tail were generated by cloning a recursive PCR product generated using four overlapping oligonucleotides (Supplementary Table 6 ) into a pQE-derivative vector 77 (Supplementary Table 7 ).…”