Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p

Abstract

The nucleolar protein Nep1 and its human homologue were previously shown to be involved in the maturation of 18S rRNA and to interfere directly or indirectly with a methylation reaction. Here, we report that the loss-of-function mutation Deltasnr57 and multicopy expression of the ribosomal 40S subunit protein 19 (Rps19p) can partially suppress the Saccharomyces cerevisiae Deltanep1 growth defect. SnR57 mediates 2'-O-ribose-methylation of G(1570) in the 18S rRNA. By performing a three-hybrid screen, we isolated several short RNA sequences with strong binding affinity to Nep1p. All isolated RNAs shared a six-nucleotide consensus motif C/UUCAAC. Furthermore, one of the isolated RNAs exactly corresponded to nucleotides 1553-1577 of the 18S rRNA, which includes G(1570), the site of snR57-dependent 18S rRNA methylation. From protein-protein crosslink data and the cryo-EM map of the S. cerevisiae small ribosomal subunit, we suggest that Rps19p is localized in close vicinity to the Nep1p 18S rRNA binding site. Our results suggest that Nep1p binds adjacent to helix 47 of the 18S rRNA and possibly supports the association of Rps19p to pre-ribosomal particles.

Powering through ribosome assembly

“…snR57 and Rps19 are predicted to bind near each other in the 39 domain of 18S rRNA, which also contains an RNA sequence identified in a three-hybrid screen with Nep1 ( Fig. 3; Buchhaupt et al 2006). Together, these results suggest a model in which Nep1 competes with snR57, to facilitate the incorporation of Rps19, possibly by effecting a conformational switch in the rRNA structure.…”

“…The role of the putative methylase Emg1/Nep1 is more nebulous. A lethal mutation in Nep1 can be rescued by deletion of the snoRNA snR57, or by overexpression of the ribosomal protein Rps19 (Buchhaupt et al 2006). snR57 and Rps19 are predicted to bind near each other in the 39 domain of 18S rRNA, which also contains an RNA sequence identified in a three-hybrid screen with Nep1 ( Fig.…”

Diamond-Blackfan anemia: erythropoiesis lost in translation

“…62 As most if not all RPs, RPS19 interacts with rRNA, and it is predicted to bind to helix 45 of the 18S rRNA. 56 Although this RPS19-18S rRNA interaction is not yet physically confirmed, RPS19 has been found to play a pivotal role in 18S rRNA maturation and 40S ribosomal subunit assembly. In RPS19-deficient yeast cells and human HeLa cells pre-40S subunits containing incompletely processed 18S rRNA accumulate in the nucleus, with a corresponding decrease in the amount of mature 40S subunits in the cytoplasm.…”

“…55 Perhaps a more interesting binding partner is the nucleolar ribosome biogenesis protein, Nep1p, that binds 18S rRNA and possibly interacts with RPS19 during formation of preribosomal 40S particles in yeast. 56 Another interesting RPS19-interacting protein is fibroblast growth factor-2, an important regulator of rRNA transcription, 57 which interacts with a pool of free RPS19 in vitro. 58 This suggests that free extraribosomal RPS19 might play a role in the signaling pathways that regulate rRNA transcription.…”

The archaeal COG1901/DUF358 SPOUT-methyltransferase members, together with pseudouridine synthase Pus10, catalyze the formation of 1-methylpseudouridine at position 54 of tRNA

“…This enzyme belongs to Cluster of Orthologous Group (COG) 1756 (Tatusov et al 2003) and converts C into m 1 C in small synthetic fragments of 8, 9, or 11 nucleotides in length (GAUUCAACGCC, where the second of the two adjacent Us is C) (Wurm et al 2010). This motif corresponds to the sequence in helix 35 of SSU rRNA of S. cerevisiae as well as of M. jannaschii, where Nep1 tightly binds (Buchhaupt et al 2006). Nep1 is responsible for the methylation step in the formation of the hypermodified 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine (m 1 acp 3 C) found at position 1191 in helix 35 of yeast 18S rRNA and also has a role in ribosome assembly (Fig.…”