Rob W. van Nues

Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits

In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of Fun12, the yeast homolog of cytoplasmic translation initiation factor eIF5b. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associated with Fun12 and formed 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together.

The Spatial-Functional Coupling of Box C/D and C′/D′ RNPs Is an Evolutionarily Conserved Feature of the Eukaryotic Box C/D snoRNP Nucleotide Modification Complex

ABSTRACT Box C/D ribonucleoprotein particles guide the 2′-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers, assembled around the C/D and C′/D′ motifs in the box C/D RNA. The C/D and C′/D′ RNPs of the archaeal snoRNA-like RNP (sRNP) are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The C/D RNP guided 2′-O-methylation when the C′/D′ motif was either mutated or ablated. In contrast, the C′/D′ RNP was inactive as an independent complex. Additional experiments demonstrated that the internal C′/D′ RNP is spatially coupled to the terminal box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D and C′/D′ motifs. Therefore, the spatial-functional coupling of box C/D and C′/D′ RNPs is an evolutionarily conserved feature of both archaeal and eukaryotic box C/D RNP complexes.

Roles for Srp72p in assembly, nuclear export and function of the signal recognition particle

Co-translational protein targeting to the endoplasmic reticulum is catalysed by the signal recognition particle, a conserved ribonucleoprotein. Key activities of SRP – signal sequence binding, and inhibition of ribosomal translation elongation – require interactions of SRP with distant locations on the ribosome. A heterodimer of Srp72p and Srp68p localise to the central portion of the SRP complex, and may co-ordinate its activities. A series of mutations within Srp72p were examined individually, in combination and in the presence of mutations within SRP RNA. In this analysis mutations within Srp72p fell into two groups, identifying separate interactions/functions of the protein. Much of Srp72p is predicted to be alpha helical tetratricopeptide repeat motifs, with the C-terminus forming a separate unstructured region. Mutations towards the C-terminal end of the alpha helical region reveal a specific genetic interaction with a conserved motif in the central helix of SRP RNA. In contrast, mutations within the C-terminal region of Srp72p have genetic interactions across the RNA. Many mutant combinations impaired function rather than inhibiting assembly of SRP. However, one specific combination of Srp72p and SRP RNA mutations led to accumulation of pre-SRP in the nucleus. We conclude that that Srp72p has at least two functions that are individually redundant and that the conformation of the complex is critical for efficient completion of its biogenesis.

Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate.

Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins.

Unusual C'/D' motifs enable box C/D snoRNPs to modify multiple sites in the same rRNA target region

Abstract Eukaryotic box C/D small nucleolar (sno)RNPs catalyse the site-specific 2΄-O-methylation of ribosomal RNA. The RNA component (snoRNA) contains guide regions that base-pair with the target site to select the single nucleotide to be modified. The terminal C/D and internal C΄/D΄ motifs in the snoRNA, adjacent to the guide region, function as binding sites for the snoRNP proteins including the enzymatic subunit fibrillarin/Nop1. Four yeast snoRNAs are unusual in that they are predicted to methylate two nucleotides in a single target region. In each case, the internal C΄/D΄ motifs from these snoRNAs differ from the consensus. Our data indicate that the C΄/D΄ motifs in snR13, snR48 and U18 form two alternative structures that lead to differences in the position of the proteins bound to this motif. We propose that each snoRNA forms two different snoRNPs, subtly different in how the proteins are bound to the C΄/D΄ motif, leading to 2΄-O-methylation of different nucleotides in the target region. For snR48 and U18, the unusual C΄/D΄ alone is enough for the modification of two nucleotides. However, for the snR13 snoRNA the unusual C΄/D΄ motif and extra base-pairing, which stimulates rRNA 2΄-O-methylation, are both critical for multiple modifications in the target region.