Identification of Genes That Function in the Biogenesis and Localization of Small Nucleolar RNAs in Saccharomyces cerevisiae

Abstract

Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.After synthesis in the nucleus, all RNA species undergo a series of maturation steps and transport from the site of synthesis to the site of action, and it is increasingly clear that RNA biogenesis and trafficking are closely linked (54). Whether they are destined to function in the cytoplasm (e.g., rRNA, tRNA, mRNA) or the nucleus (e.g., snRNAs or snoRNAs), all RNAs undergo intranuclear trafficking. It is well established that interactions between trans-acting factors and specific features of RNA substrates can govern which transport pathways will be utilized (38,59,73). However, little is known about the intranuclear trafficking factors and how they affect the molecular movement of RNAs within the nucleus.Small nucleolar RNAs (snoRNAs) provide excellent models to study intranuclear RNA transport. snoRNAs remain within the nucleus, where they undergo biogenesis including covalent alterations (e.g., 5Ј cap hypermethylation and 3Ј-end processing), as well as assembly with specific proteins into stable and functional RNP complexes (snoRNPs) (40,54,70,84). Ultimately, snoRNAs are targeted to nucleoli, where they function in rRNA maturation.The numerous snoRNA species fall into two structurally and functionally distinct classes called the box C/D and box H/ACA snoRNAs (40,54,70,84). Species of both RNA classes interact with pre-rRNA (via base pairing), mainly to guide site-specific nucleotide modification. The majority of box C/D snoRNAs orchestrate 2Ј-O-methylation (12,41,65,89), while the bulk of the box H/ACA RNA species guide pseudouridylation (21, 64). A few RNAs of both classes (e.g., box C/D snoRNA U3 and box H/ACA snoRNA U17 [vertebrates]/snR30 [yeast]) are critical for promoting specific endonucleolytic cleavages of pre-rRNA transcripts ...

A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis

“…Consistent with our microarray-based analysis of Zuo1 function (Fig. 7 B), Rrp1 is involved in 27S rRNA processing (Horsey et al, 2004), Rrp15 is involved in 35S rRNA processing (De Marchis et al, 2005), and Nop4 facilitates localization to the nucleolus of the U3 snoRNA, which is required for rRNA processing of both 35S and 27S rRNA (Sun and Woolford, 1994;Qiu et al, 2008). Thus, our results resonate with several genetic and biochemical observations.…”

Mutations in the Saccharomyces cerevisiae Kinase Cbk1p Lead to a Fertility Defect That Can Be Suppressed by the Absence of Brr1p or Mpt5p (Puf5p), Proteins Involved in RNA Metabolism

“…No clear unified picture is immediately evident from all these interactions, but there are certain parallels that draw these proteins together. Cbk1p, Ssd1p, and Mpt5p have all been linked to the maintenance of cell integrity; Ssd1p, Brr1p, and Mpt5p are all involved in RNA metabolism; and recently Tao3p, another RAM component, has been implicated in snoRNA biogenesis (Qiu et al 2008). Brr1p and Mpt5p are known to interact with the Ccr4-Not complex that mediates 59-39 mRNA deadenylation and Cbk1p is also linked to this complex as it is a two-hybrid partner of one of the subunits (Not3p) (Racki et al 2000).…”

The Yeast LATS/Ndr Kinase Cbk1 Regulates Growth via Golgi-dependent Glycosylation and Secretion

“…In support, Cbk1 binds Ssd1 by two-hybrid (Racki et al, 2000). Moreover, the RAM network protein Tao3 was recently shown to be involved in small nucleolar RNA maturation and localization (Qiu et al, 2008). Further work is necessary to elucidate the mechanism of Ssd1 function with regard to Cbk1 and RAM network function.…”