E. Stuart Maxwell

The nucleolar snRNAs: catching up with the spliceosomal snRNAs

Despite their early discovery, research into the small RNAs associated with the eukaryotic nucleolus (snoRNAs) has lagged behind that of their cousins, the small nuclear RNAs which are known to function in mRNA splicing (spliceosomal snRNAs). Recent progress has now shown that the snoRNAs also occupy a vital niche in the RNA world, participating in the processing of ribosomal RNA. Like the spliceosomal snRNAs, the snoRNAs exist as ribonucleoprotein (RNP) particles which appear to assemble into a large multi-RNA RNP complex for pre-rRNA maturation.

Efficient RNA 2′‐ O ‐methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C′/D′ RNPs

Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide‐specific 2′‐O‐methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C′/D′ RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C′/D′ motifs has demonstrated that the terminal box C/D RNP is the minimal methylation‐competent particle. However, efficient ribonucleotide 2′‐O‐methylation requires that both the box C/D and C′/D′ RNPs function within the full‐length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C′/D′ motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA‐binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles.

Mouse U14 snRNA is a processed intron of the cognate hsc70 heat shock pre-messenger RNA

U14 snRNA is a small nucleolar RNA species essential for eukaryotic pre-rRNA processing. We have previously shown that the mouse U14 snRNA genes are positioned within introns 5, 6, and 8 on the coding strand of the constitutively expressed cognate hsc70 heat shock gene. This genomic organization suggested the possibility that U14 snRNAs are transcribed as part of the hsc70 pre-mRNA and then excised from the intron to yield mature U14 snRNA species. To test this hypothesis directly, we have microinjected Xenopus oocytes with hsc70 pre-mRNA transcripts possessing intron 5 and the encoded U14 snRNA sequence. Processing results demonstrate that, in addition to the splicing of upstream and downstream exons, a mature 87 nt U14 snRNA is excised from the intron. Accurate excision of U14 snRNA from hsc70 intron 5 can occur in the absence of splicing. These results demonstrate a biosynthetic pathway for an snRNA species and provide a novel example of a eukaryotic pre-mRNA intron that is processed to produce a stable, biologically functional RNA species.

A Dimeric Structure for Archaeal Box C/D Small Ribonucleoproteins

Seeing Double A particular set of ubiquitous small (nucleolar) ribonucleoproteins are important for optimal ribosome function and protein synthesis. Bleichert et al. (p. 1384) used electron microscopy and single-particle analysis to investigate the structure of an archaeal version that contains the small RNA (sRNA) and all the associated core proteins. Unexpectedly, this ribonucleoprotein is a homodimer, formed of two sRNAs and four copies of each of the core proteins. This dimer is likely to be the enzymatically active form, as mutations disrupting di-sRNP formation inhibited activity. Electron microscopy and single-particle analysis show that a small nuclear ribonucleoprotein forms a dimeric structure. Methylation of ribosomal RNA (rRNA) is required for optimal protein synthesis. Multiple 2′-O-ribose methylations are carried out by box C/D guide ribonucleoproteins [small ribonucleoproteins (sRNPs) and small nucleolar ribonucleoproteins (snoRNPs)], which are conserved from archaea to eukaryotes. Methylation is dictated by base pairing between the specific guide RNA component of the sRNP or snoRNP and the target rRNA. We determined the structure of a reconstituted and catalytically active box C/D sRNP from the archaeon Methanocaldococcus jannaschii by single-particle electron microscopy. We found that archaeal box C/D sRNPs unexpectedly formed a dimeric structure with an alternative organization of their RNA and protein components that challenges the conventional view of their architecture. Mutational analysis demonstrated that this di-sRNP structure was relevant for the enzymatic function of archaeal box C/D sRNPs.

Nucleotide sequences of Cyanophora paradoxa cellular and cyanelle-associated 5S ribosomal RNAs: the cyanelle as a potential intermediate in plastid evolution.

SummaryThe 5S ribosomal RNAs from the cell cytoplasm and cyanelle (photosynthetic organelle) ofCyanophora paradoxa have been isolated and sequenced. The cellular and cyanelle 5S rRNAs were 119 and 118 nucleotides in length, respectively. Both RNAs exhibited typical 5S secondary structure, but the primary sequence of the cellular species was clearly eukaryotic in nature, while that of the organellar species was prokaryotelike. The primary sequence of the cyanellar 5S rRNA was most homologous to cyanobacterial 5S sequences, yet possessed secondary-structural features characteristic of higher-plant chloroplast 5S rRNAs. Both sequence comparison and structural analysis indicated an evolutionary position for cyanelle 5S rRNA intermediate between blue-green alga and chloroplast 5S rRNAs.

Electrophoretic Mobility Shift Assay for Characterizing RNA–Protein Interaction

Electrophoretic mobility shift assay, or EMSA, is a well-established technique for separating macromolecules under native conditions based on a combination of shape, size, and charge. The use of EMSA can provide both general and specific information concerning the interaction between two macromolecules such as RNA and protein. Here we present a protocol for the practical use of EMSA to assess protein-RNA interactions and ribonucleoprotein (RNP) assembly. The conceptual framework of the assay is discussed along with a step-by-step procedure for the binding of archaeal ribosomal protein L7Ae to a box C/D sRNA. Potential pitfalls and common mistakes to avoid are emphasized with technical tips and a notes section. This protocol provides a starting point for the design and implementation of EMSA in studying a wide variety of RNP complexes.

Evidence for a Competitive‐Displacement Model for the initiation of protein synthesis involving the intermolecular hybridization of 5 S rRNA, 18 S rRNA and mRNA

We have previously shown that a 5′‐terminal region of mouse 5 S rRNA can base‐pair in vitro with two distinct regions of 18 S rRNA. Further analysis reveals that these 5 S rRNA‐complementary sequences in 18 S rRNA also exhibit complementarity to the Kozak consensus sequence surrounding the mRNA translational start site. To test the possibility that these 2 regions in 18 S rRNA may be involved in mRNA binding and translational initiation, we have tested, using an in vitro translation system, the effects of DNA oligonucleotides complementary to these 18 S rRNA sequences on protein synthesis. Results show that an oligonucleotide complementary to one 18 S rRNA region does inhibit translation at the step of initiation. We propose a Competitive‐Displacement Model for the initiation of translation involving the intermolecular base‐pairing of 5 S rRNA 18 S rRNA and mRNA.

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.

A role for hydrophobicity in a Diels–Alder reaction catalyzed by pyridyl-modified RNA

New classes of RNA enzymes or ribozymes have been obtained by in vitro evolution and selection of RNA molecules. Incorporation of modified nucleotides into the RNA sequence has been proposed to enhance function. DA22 is a modified RNA containing 5-(4-pyridylmethyl) carboxamide uridines, which has been selected for its ability to promote a Diels–Alder cycloaddition reaction. Here, we show that DA_TR96, the most active member of the DA22 RNA sequence family, which was selected with pyridyl-modified nucleotides, accelerates a cycloaddition reaction between anthracene and maleimide derivatives with high turnover. These widely used reactants were not used in the original selection for DA22 and yet here they provide the first demonstration of DA_TR96 as a true multiple-turnover catalyst. In addition, the absence of a structural or essential kinetic role for Cu2+, as initially postulated, and nonsequence-specific hydrophobic interactions with the anthracene substrate have led to a reevaluation of the pyridine modifications role. These findings broaden the catalytic repertoire of the DA22 family of pyridyl-modified RNAs and suggest a key role for the hydrophobic effect in the catalytic mechanism.

Structurally Conserved Nop56/58 N-terminal Domain Facilitates Archaeal Box C/D Ribonucleoprotein-guided Methyltransferase Activity

Background: Box C/D RNPs direct site-specific 2′-O-methylation of rRNA. Results: The Nop56/58 and fibrillarin core proteins establish a very stable dimer with Nop56/58 contributing to methyltransferase activity. Conclusion: The Nop56/58 core protein plays a role not only in RNP assembly, but also methyltransferase activity. Significance: Our observations reveal a novel role for the Nop56/58 core protein in box C/D RNP function. Box C/D RNA-protein complexes (RNPs) guide the 2′-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C′/D′ RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity.