Essential role of NbNOG_1 in ribosomal RNA processing

Nucleolar GTP-binding protein 1(NOG_1) is a highly conserved GTPase first reported in Trypanosoma as required for ribosome biogenesis. We characterized NbNOG_1, a Nicotiana benthamiana NOG_1 ortholog sharing more than 45% amino acid identity with Trypanosoma,yeast, and human NOG_1. N. benthamiana plants silenced for NbNOG_1 were stunted and produced sterile flowers.NbNOG_1 is functionally interchangeable with yeast NOG_1(ScNOG_1), rescuing yeast lethality caused by loss of ScNOG_1. Finally, NbNOG_1 silencing caused overaccumulation of pre-rRNA processing intermediates,and concomitant loss of mature rRNAs. Collectively,these data support a role for NbNOG_1 in ribosomal RNA processing.     

A conditional lethal mutant of Escherichia coli which affects the processing of ribosomal RNA.

A temperature-sensitive mutant strain was isolated from an RNase III-(rnc) strain of Escherichia coli. At the permissive temperature it behaves like the parental strain, but at the nonpermissive temperature it fails to produce normal levels of 23 S and 5 S rRNA, while instead the 25 S rRNA species becomes very prominent. (The 25 S molecule appears in rnc cells and contains 23 S rRNA sequences). When an rnc+ mutation was introduced to such a strain, or when the rnc mutation was replaced by an rnc+ allele, the strain remained temperature-sensitive. At the permissive temperature such strains synthesized rRNA like any other E. coli strain, but at the nonpermissive temperature they remained unable to synthesize normal levels of 5 S rRNA, and instead a larger molecule was accumulated. The simplest interpretation of theses findings is that the mutant strain contains a temperature-sensitive processing endoribonuclease, RNase E, which normally introduces a cut in the growing rRNA chain somewhere between the 23 S and the 5 S rRNA cistrons. These findings help also to explain the nature and origin of the various rRNA species observed in RNase III- cells and add to our understanding of processing of ribosomal RNA in normal cells of Escherichia coli.     

APP processing and synaptic plasticity in presenilin-1 conditional knockout mice

We have developed a presenilin-1 (PS1) conditional knockout mouse (cKO), in which PS1 inactivation is restricted to the postnatal forebrain. The PS1 cKO mouse is viable and exhibits no gross abnormalities. The carboxy-terminal fragments of the amyloid precursor protein differentially accumulate in the cerebral cortex of cKO mice, while generation of beta-amyloid peptides is reduced. Expression of Notch downstream effector genes, Hes1, Hes5, and Dll1, is unaffected in the cKO cortex. Although basal synaptic transmission, long-term potentiation, and long-term depression at hippocampal area CA1 synapses are normal, the PS1 cKO mice exhibit subtle but significant deficits in long-term spatial memory. These results demonstrate that inactivation of PS1 function in the adult cerebral cortex leads to reduced Abeta generation and subtle cognitive deficits without affecting expression of Notch downstream genes.     

Evidence for an RNA pseudoknot loop-helix interaction essential for efficient -1 ribosomal frameshifting.

RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2.     

Nol9 is a novel polynucleotide 5'-kinase involved in ribosomal RNA processing

In a cell, an enormous amount of energy is channelled into the biogenesis of ribosomal RNAs (rRNAs). In a multistep process involving a large variety of ribosomal and non-ribosomal proteins, mature rRNAs are generated from a long polycistronic precursor. Here, we show that the non-ribosomal protein Nol9 is a polynucleotide 5'-kinase that sediments primarily with the pre-60S ribosomal particles in HeLa nuclear extracts. Depletion of Nol9 leads to a severe impairment of ribosome biogenesis. In particular, the polynucleotide kinase activity of Nol9 is required for efficient generation of the 5.8S and 28S rRNAs from the 32S precursor. Upon Nol9 knockdown, we also observe a specific maturation defect at the 5' end of the predominant 5.8S short-form rRNA (5.8S(S)), possibly due to the Nol9 requirement for 5'>3' exonucleolytic trimming. In contrast, the endonuclease-dependent generation of the 5'-extended, minor 5.8S long-form rRNA (5.8S(L)) is largely unaffected. This is the first report of a nucleolar polynucleotide kinase with a role in rRNA processing.     

Exoribonuclease R in Pseudomonas syringae is essential for growth at low temperature and plays a novel role in the 3' end processing of 16 and 5 S ribosomal RNA

The (3'-->5') exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the cold-adapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4 degrees C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4 degrees C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4 degrees C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3' ends. This suggests a novel role for RNase R in the rRNA 3' end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4 degrees C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the cold-adapted Antarctic P. syringae.     

A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight,inducible transgene expression

Background

One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression.

Results

We constructed a single adenoviral vector carrying in its E1 region a novel “all-in-one” Tet-On system with an autoregulatory loop. This system had improved Dox-inducible gene expression in terms of low basal expression, high induced expression and high responsiveness to Dox. To our knowledge, this is the first reported adenovirus-based, all-in-one Tet-On system with an autoregulatory loop inserted into a single region of adenoviral genome. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). The adenovirus that expressed soluble TRAIL under the control of this novel Tet-On system showed tumor-derived cells inhibitory activity in SW480 cells only under induced conditions.

Conclusions

Our novel, single adenoviral vector carrying in its E1 region an all-in-one Tet-On system with an autoregulatory loop displayed tight regulation of transgene expression in vitro. This system has great potential for a variety of applications, including gene therapy and the study of gene function.     

5S ribosomal RNA is contained within a 25 S ribosomal RNA that accumulates in mutants of Escherichia coli defective in processing of ribosomal RNA.

A temperature-sensitive mutant strain of Escherichia coli defective in two RNA processing enzymes, RNase III and RNase E (rnc. rne), fails to produce normal levels of 23 S and 5 S rRNA at the non-permissive temperature. Instead, a molecule larger than 23 S is produced. This molecule, designated 25 S rRNA, can be processed in vitro to produce p5 rRNA. These findings further our understanding of the overall processing events of ribosomal RNA which take place in the bacterial cell.     

Evidence for involvement of NFBP in processing of ribosomal RNA

Ribosomal RNA (rRNA) in vertebrates is initially transcribed as a single 47S precursor which is modified by the addition of 2'-O-methyl ribose moieties, pseudouridines, and methyl groups, followed by cleavage at several sites to produce the mature 28S, 18S, and 5.8S rRNAs. Cleavage of the rRNA precursor to generate the 18S rRNA is mediated by a ribonucleoprotein (RNP) complex termed the processome containing U3, a box C/D small nucleolar RNA (snoRNA), and at least 28 cellular proteins. We previously identified a novel human RNA binding protein, NF-kappaB binding protein (NFBP), which is the human homolog of Rrp5p, a protein component of the yeast U3 processome. Here, we show that NFBP colocalizes with and coprecipitates U3 in the nucleolus. We also demonstrate that NFBP is essential for the generation of 18S rRNA as maturation of the 18S rRNA is repressed in the absence of NFBP. Using Northern blot analyses, we further show that NFBP is specifically necessary for cleavages at sites A0, 1, and 2, as unprocessed intermediate forms of rRNA accumulated in the absence of NFBP.