Mammalian DEAD box protein Ddx51 acts in 3' end maturation of 28S rRNA by promoting the release of U8 snoRNA.

Biogenesis of eukaryotic ribosomes requires a number of RNA helicases that drive molecular rearrangements at various points of the assembly pathway. While many ribosome synthesis factors are conserved among all eukaryotes, certain features of ribosome maturation, such as U8 snoRNA-assisted processing of the 5.8S and 28S rRNA precursors, are observed only in metazoan cells. Here, we identify the mammalian DEAD box helicase family member Ddx51 as a novel ribosome synthesis factor and an interacting partner of the nucleolar GTP-binding protein Nog1. Unlike any previously studied yeast helicases, Ddx51 is required for the formation of the 3' end of 28S rRNA. Ddx51 binds to pre-60S subunit complexes and promotes displacement of U8 snoRNA from pre-rRNA, which is necessary for the removal of the 3' external transcribed spacer from 28S rRNA and productive downstream processing. These data demonstrate the emergence of a novel factor that facilitates a pre-rRNA processing event specific for higher eukaryotes.

Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells.

Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.

The 5' external transcribed spacer in mouse ribosomal RNA contains two cleavage sites.

The 5' external transcribed spacer (5'ETS) is critical for 18S rRNA formation and is the longest noncoding region in a ribosomal RNA transcript. Here we show that processing in mouse 5'ETS involves two cleavage events. Processing at site A' corresponds to the previously described "primary cleavage," which precedes other processing steps. Processing at the novel site A0 occurs 1 kb downstream from A' yielding two new rRNA precursors: 43S and 29S. The excised 5'-A' and A'-A0 fragments are rapidly degraded under normal conditions. Depletion of the exosome component EXOSC10/PM-Scl100 (ortholog of yeast Rrp6p) results in a strong accumulation of the A'-A0 spacer fragment in mouse cells. We discuss the finding of a second processing site in mammalian 5'ETS in relation to the involvement of the U3 snoRNA in pre-rRNA processing and present a revised map of the mouse 18S rRNA processing pathway.

Assays for ribosomal RNA processing and ribosome assembly.

The synthesis of ribosomes is a major metabolic activity critical for cell growth and homeostasis. Understanding the mechanisms of ribosome biogenesis has important implications for studying both protein synthesis and cell cycle control. This unit describes several techniques for the analysis of rRNA maturation and ribosome assembly adapted for mammalian cells. Metabolic labeling of rRNA and hybridization analysis of precursors can be used to assess changes in rRNA processing that occur under experimental conditions of interest. Separation of preribosomal particles by sucrose gradient centrifugation is suitable for the analysis of proteins associated with preribosomes during their assembly and maturation in the cell nucleus.

Restricting conformational flexibility of the switch II region creates a dominant-inhibitory phenotype in Obg GTPase Nog1.

Nog1 is a conserved eukaryotic GTPase of the Obg family involved in the biogenesis of 60S ribosomal subunits. Here we report the unique dominant-inhibitory properties of a point mutation in the switch II region of mouse Nog1; this mutation is predicted to restrict conformational mobility of the GTP-binding domain. We show that although the mutation does not significantly affect GTP binding, ectopic expression of the mutant in mouse cells disrupts productive assembly of pre-60S subunits and arrests cell proliferation. The mutant impairs processing of multiple pre-rRNA intermediates, resulting in the degradation of the newly synthesized 5.8S/28S rRNA precursors. Sedimentation analysis of nucleolar preribosomes indicates that defective Nog1 function inhibits the conversion of 32S pre-rRNA-containing complexes to a smaller form, resulting in a drastic accumulation of enlarged pre-60S particles in the nucleolus. These results suggest that conformational changes in the switch II element of Nog1 have a critical importance for the dissociation of preribosome-bound factors during intranucleolar maturation and thereby strongly influence the overall efficiency of the assembly process.

The GCR1, GPA1, PRN1, NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis.

Different classes of biotic (e.g. plant hormones) and abiotic (e.g. different wavelengths of light) signals act through specific signal transduction mechanisms to coordinate higher plant development. While a great deal of progress has been made, full signal transduction chains have not yet been described for most blue light- or abscisic acid-mediated events. Based on data derived from T-DNA insertion mutants and yeast (Saccharomyces cerevisiae) two-hybrid and coprecipitation assays, we report a signal transduction chain shared by blue light and abscisic acid leading to light-harvesting chlorophyll a/b-binding protein expression in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. The chain consists of GCR1 (the sole Arabidopsis protein coding for a potential G-protein-coupled receptor), GPA1 (the sole Arabidopsis Galpha-subunit), Pirin1 (PRN1; one of four members of an iron-containing subgroup of the cupin superfamily), and a nuclear factor Y heterotrimer comprised of A5, B9, and possibly C9. We also demonstrate that this mechanism is present in imbibed seeds wherein it affects germination rate.

Physical and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis.

Molecular mechanisms of mammalian ribosome biogenesis remain largely unexplored. Here we develop a series of transposon-derived dominant mutants of Pes1, the mouse homolog of the zebrafish Pescadillo and yeast Nop7p implicated in ribosome biogenesis and cell proliferation control. Six Pes1 mutants selected by their ability to reversibly arrest the cell cycle also impair maturation of the 28S and 5.8S rRNAs in mouse cells. We show that Pes1 physically interacts with the nucleolar protein Bop1, and both proteins direct common pre-rRNA processing steps. Interaction with Bop1 is essential for the efficient incorporation of Pes1 into nucleolar preribosomal complexes. Pes1 mutants defective for the interaction with Bop1 lose the ability to affect rRNA maturation and the cell cycle. These data show that coordinated action of Pes1 and Bop1 is necessary for the biogenesis of 60S ribosomal subunits.

The Arabidopsis cupin domain protein AtPirin1 interacts with the G protein alpha-subunit GPA1 and regulates seed germination and early seedling development.

Heterotrimeric G proteins are implicated in diverse signaling processes in plants, but the molecular mechanisms of their function are largely unknown. Finding G protein effectors and regulatory proteins can help in understanding the roles of these signal transduction proteins in plants. A yeast two-hybrid screen was performed to search for proteins that interact with Arabidopsis G protein alpha-subunit (GPA1). One of the identified GPA1-interacting proteins is the cupin-domain protein AtPirin1. Pirin is a recently defined protein found because of its ability to interact with a CCAAT box binding transcription factor. The GPA1-AtPirin1 interaction was confirmed in an in vitro binding assay. We characterized two atpirin1 T-DNA insertional mutants and established that they display a set of phenotypes similar to those of gpa1 mutants, including reduced germination levels in the absence of stratification and an abscisic acid-imposed delay in germination and early seedling development. These data indicate that AtPirin1 likely functions immediately downstream of GPA1 in regulating seed germination and early seedling development.