Brenda A. Peculis

Xenopus U8 snoRNA Binding Protein Is a Conserved Nuclear Decapping Enzyme

U8 snoRNP is required for accumulation of mature 5.8S and 28S rRNA in vertebrates. We are identifying proteins that bind U8 RNA with high specificity to understand how U8 functions in ribosome biogenesis. Here, we characterize a Xenopus 29 kDa protein (X29), which we previously showed binds U8 RNA with high affinity. X29 and putative homologs in other vertebrates contain a NUDIX domain found in MutT and other nucleotide diphosphatases. Recombinant X29 protein has diphosphatase activity that removes m(7)G and m(227)G caps from U8 and other RNAs in vitro; the putative 29 kDa human homolog also displays this decapping activity. X29 is primarily nucleolar in Xenopus tissue culture cells. We propose that X29 is a member of a conserved family of nuclear decapping proteins that function in regulating the level of U8 snoRNA and other nuclear RNAs with methylated caps.

Xenopus LSm Proteins Bind U8 snoRNA via an Internal Evolutionarily Conserved Octamer Sequence

ABSTRACT U8 snoRNA plays a unique role in ribosome biogenesis: it is the only snoRNA essential for maturation of the large ribosomal subunit RNAs, 5.8S and 28S. To learn the mechanisms behind the in vivo role of U8 snoRNA, we have purified to near homogeneity and characterized a set of proteins responsible for the formation of a specific U8 RNA-binding complex. This 75-kDa complex is stable in the absence of added RNA and binds U8 with high specificity, requiring the conserved octamer sequence present in all U8 homologues. At least two proteins in this complex can be cross-linked directly to U8 RNA. We have identified the proteins as Xenopus homologues of the LSm (like Sm) proteins, which were previously reported to be involved in cytoplasmic degradation of mRNA and nuclear stabilization of U6 snRNA. We have identified LSm2, -3, -4, -6, -7, and -8 in our purified complex and found that this complex associates with U8 RNA in vivo. This purified complex can bind U6 snRNA in vitro but does not bind U3 or U14 snoRNA in vitro, demonstrating that the LSm complex specifically recognizes U8 RNA.

Identification of a U8 snoRNA-specific binding protein.

Eukaryotic nucleoli contain a large and diverse population of small nucleolar ribonucleoprotein particles (snoRNPs) that play diverse and essential roles in ribosome biogenesis. We previously demonstrated that U8 snoRNP is essential for processing of both 5.8 and 28 S rRNA. The RNA component of the U8 RNP particle is necessary but not sufficient for processing. Using an electrophoretic mobility sift assay, we enriched for U8-specific binding proteins fromXenopus ovary extracts. UV cross-linking reactions with partially purified fractions implicated a 29-kDa protein directly binding to U8 RNA. This protein interacted specifically and with high affinity with U8 snoRNA; it did not bind other snoRNAs and is probably not a common component of all snoRNPs. This is the first report of a protein component specific to an snoRNP essential for processing of the large ribosomal subunit in vertebrates.

snoRNA nuclear import and potential for cotranscriptional function in pre-rRNA processing.

Several snoRNAs are essential for the sequence of cleavage events required to produce the mature forms of 18S, 5.8S, and 28S rRNA from the large precursor molecule. In the absence of U22, mature 18S rRNA fails to accumulate; U8 snoRNA is essential for accumulation of both 5.8S and 28S rRNA. The mechanisms by which snoRNAs facilitate these cleavage events is not known and might include direct cleavage or assisting the rate or efficiency of ribosome assembly. To learn more about the mechanisms of snoRNA-mediated pre-rRNA processing, an examination of the kinetics of pre-rRNA processing in Xenopus oocytes was undertaken. Correct pre-rRNA processing can be restored in snoRNA-depleted oocytes following cytoplasmic injection of the corresponding in vitro-synthesized snoRNA. Analysis of the kinetics of pre-rRNA processing in these snoRNA-rescue experiments demonstrated that the rate of accumulation of mature rRNAs was slower than that seen in untreated oocytes. The snoRNAs were imported into the nucleus at a rate and overall efficiency less than that of U1 snRNA, used as a control for import. However, sufficient levels of snoRNA were present in the nucleus to yield a functional phenotype (rescue of rRNA processing) several hours before the snoRNAs were directly detectable in the nucleus via autoradiography. This indicated that very low amounts of the snoRNA in the nucleus were sufficient for rescue. Finally, transcriptional inhibitors were used to separate transcription and processing. Failure to rescue snoRNA-mediated processing of pre-accumulated precursors is consistent with a scenario in which U8 and U22 must be present during transcription of pre-rRNA.

Ribosomal RNA: Small nucleolar RNAs make their mark

Small nucleolar RNAs direct the location of certain methylations in ribosomal RNA by direct base pairing; although evolutionarily conserved, the physiological significance of these modifications remains unclear.

Metal Determines Efficiency and Substrate Specificity of the Nuclear NUDIX Decapping Proteins X29 and H29K (Nudt16)

The Xenopus X29 protein was identified by its high affinity binding to U8 small nucleolar RNA, a small nucleolar RNA required for ribosome biogenesis. X29 and its human homologue H29K (Nudt16) are nuclear nucleoside diphosphatase proteins localized within foci in the nucleolus and nucleoplasm. These proteins can remove m7G and m227G caps from RNAs, rendering them substrates for 5′-3′ exonucleases for degradation in vivo. Here, a more complete characterization of these metal-dependent decapping proteins demonstrates that the metal identity determines both the efficiency of decapping and the RNA substrate specificity. In Mg+2 the proteins hydrolyze the 5′ cap from only one RNA substrate: U8 small nucleolar RNA. However, in the presence of Mn+2 or Co+2 all RNAs are substrates and the decapping efficiency is higher. The x-ray crystal structure of X29 facilitated structure-based mutagenesis. Mutation of single amino acids coordinating metal in the active site yielded mutant proteins confirming essential residues. In vitro assays with purified components are consistent with a lack of protein turnover, apparently due to an inability of the protein to release the decapped RNA, implicating critical in vivo interacting factors. Collectively, these studies indicate that the metal that binds the X29/H29K proteins in vivo may determine whether these decapping proteins function solely as a negative regulator of ribosome biogenesis or can decap a wider variety of nuclear-limited RNAs. With the potential broader RNA substrate specificity, X29/H29K may be the nuclear counterparts of the cytoplasmic decapping machinery, localized in specialized bodies involved in RNA decay.

Ribosome Biogenesis: Ribosomal RNA Synthesis as a Package Deal

Ribosome biogenesis encompasses a complicated series of events involving hundreds of transiently interacting components. Insight into a mechanism for coordinating some of these events may come from characterization of a functional processing complex.

RNA-binding proteins: If it looks like a sn(o)RNA…

Small nuclear RNAs are involved in splicing pre-mRNA, while small nucleolar RNAs facilitate ribosome biogenesis. But these distinct particles may have more in common than was first apparent: some of their RNA components share a common RNA binding protein, a common RNA structure and perhaps a common origin.

The U8 snoRNA gene family: identification and characterization of distinct, functional U8 genes in Xenopus

U8 snoRNA is the RNA component of a small nucleolar ribonucleoprotein (U8 snoRNP) required for accumulation of mature 5.8S and 28S rRNAs, components of the large ribosomal subunit. We have identified two putative U8 genes in Xenopus laevis. Sequence analysis of the coding regions of these two genes indicate that both differ at several positions from the previously characterized U8 RNA and that the two differ from each other. Functional analysis of these genes indicates that both are transcribed in vivo, produce stable U8 transcripts, and are capable of facilitating pre-rRNA processing in vivo. These data demonstrate that natural sequence variation exists among the U8 snoRNA genes in Xenopus. Alignment of these three Xenopus U8 sequences with the previously described mammalian U8 homologues in mouse, rat and human has provided information about evolutionarily conserved sequence and structural elements in U8 RNA. Identification and functional characterization of these naturally occurring variants in Xenopus has helped identify regions in U8 RNA that may be critical for function.

Crystals of X29, a Xenopus laevis U8 snoRNA-binding protein with nuclear decapping activity.

Eukaryotic ribosome biosynthesis requires modification (methylation, pseudouridylation) and nucleolytic processing of precursor ribosomal RNAs in the nucleolus. The RNA components of the small nucleolar RNPs (snoRNAs) are essential for many of these events. One snoRNP, called U8, is necessary for maturation of 5.8S and 28S rRNA in vertebrates. In Xenopus laevis, U8 snoRNA was found to bind specifically and with high affinity to a protein called X29. X29 is a Nudix hydrolase, a nucleotide diphosphatase that removes the m(7)G and m(227)G caps from U8 and other RNAs. X29 requires an RNA as substrate and cap analogues are not substrates or inhibitors of cleavage. To study the determinants of X29 activity and its specificity for U8 RNA substrate, X29 was crystallized in an orthorhombic crystal form that diffracts to 2.1 A resolution.