A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase

Abstract

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the "foot" region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.

In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation

“…As snoRNA-rRNA interactions are destabilized by helicases upon binding to pre-rRNA, SPLASH analysis in yeast cells that overexpress the helicase Prp43 mutant (Bohnsack et al, 2009;Martin et al, 2002) (prp43-T123A; Figure S6D) was used to identify additional transient snoRNA-rRNA interactions that are important for rRNA biogenesis. The essential H/ACA box snR30 was previously found to be released from 18S rRNA by the Rok1 helicase and is required for 35S cleavage and release of the 18S rRNA precursor (Martin et al, 2014). In our analysis, we identified snR30-18S rRNA interactions in the Prp43 mutant but not in the wild-type ( Figure 5E), suggesting that either multiple helicases can work on the same snoRNA substrate(s) to facilitate their release from rRNA or that Prp43 is required for Rok1 to unwind snR30 from the preribosome.…”

Methylation guide RNA evolution in archaea: structure, function, and genomic organization of 110 C/D box sRNA families across sixPyrobaculumspecies

“…Nonetheless, a mismatch at the site of methylation within a conserved guide-target complementarity implies that the interaction may be beneficial but that the modification is either not needed or harmful to the function of the target RNA. Studies in yeast that use cross-linking to detect RNA-RNA interactions support this hypothesis (39,40).…”

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

“…To date, 21 RNA helicases have been suggested to act during the process of ribosome biogenesis in yeast with eight implicated in SSU maturation, ten in LSU biogenesis and three in the assembly of both subunits [20]. A wealth of research has provided information on the binding sites and potential functions of many of these RNA helicases [21][22][23][24][25][26][27][28][29][30][31][32][33], with several RNA helicases implicated in promoting the release of specific snoRNPs from early pre-ribosomal particles [21,22,26,27,30,31] and others suggested to catalyse pre-rRNA rearrangements that induce formation of mature rRNA conformations facilitating the recruitment of RPs and/or AFs [23,32,34]. However, in the case of other RNA helicases that have been found associated with pre-ribosomal particles, little is known about the timing of their association, the interactions they make within pre-ribosomes and what contribution their activity makes to the assembly process.…”

“…The most prominent example is the G-patch proteins, which contain a glycinerich domain via which they associate with and regulate the activity of the DEAH box helicases Prp43 and Prp2 [40][41][42][43][44]. During ribosome assembly, Prp43 mediates the release of a specific subset of snoRNPs from early pre-LSU particles, a function which has been linked to the G-patch protein Gno1 (Pxr1) [27,45]. In addition, Prp43 is suggested to remodel cytoplasmic pre-SSU particles to facilitate cleavage at the 3ʹ end of the 18S rRNA and the G-patch protein Pfa1 (Sqs1) has been linked to this function [27,28,46].…”

“…During ribosome assembly, Prp43 mediates the release of a specific subset of snoRNPs from early pre-LSU particles, a function which has been linked to the G-patch protein Gno1 (Pxr1) [27,45]. In addition, Prp43 is suggested to remodel cytoplasmic pre-SSU particles to facilitate cleavage at the 3ʹ end of the 18S rRNA and the G-patch protein Pfa1 (Sqs1) has been linked to this function [27,28,46]. Alongside the G-patch proteins, a family of cofactors dedicated to regulation of eIF4A-like RNA helicases and characterized by the presence of a 'middle of eIF4G' (MIF4G) domain has been described ( [47] and reviewed in [35,48]).…”