Functional significance of intermediate cleavages in the 3'ETS of the pre-rRNA from Schizosaccharomyces pombe

Pathways for the maturation of ribosomal RNAs are complex with numerous intermediate cleavage sites that are not always conserved closely in the course of evolution. Both in eukaryotes and bacteria genetic analyses and in vitro studies have strongly implicated RNase III-like enzymes in the processing of rRNA precursors. In Schizosacharomyces pombe, for example, the RNase III-like Pac1 nuclease has been shown to cleave the free 3′ETS at two known intermediate sites but, in the presence of RAC protein, the same RNA also is cleaved at the 3′-end of the 25 S rRNA sequence. In this study normal and mutant 3′ETS sequences were digested with the Pac1 enzyme to further evaluate its role in rRNA processing. Accurate cleavage at the known intermediate processing sites was dependent on the integrity of the helical structure at these sites as well as a more distal upper stem region in the conserved extended hairpin structure of the 3′ETS. The cleavage of mutant 3′ETS sequences also generally correlated with the known effects of these mutations on rRNA production, in vivo. One mutant, however, was efficiently processed in vivo but was not a substrate for the Pac1 nuclease, in vitro. In contrast, in the presence of RAC protein, the same RNA remained susceptible to Pac1 nuclease cleavage at the 3′-end of the 25 rRNA sequence, indicating that the removal of the 3′ETS does not require cleavage at the intermediate sites. These results suggest that basic maturation pathways may be less complex than previously reported raising similar questions about other intermediate processing sites, which have been identified by analyses of termini, and/or processing, in vitro.     

Processing of Bacillus subtilis small cytoplasmic RNA: evidence for an additional endonuclease cleavage site

Small cytoplasmic RNA (scRNA) of Bacillus subtilis is the RNA component of the signal recognition particle. scRNA is transcribed as a 354-nt precursor, which is processed to the mature 271-nt scRNA. Previous work demonstrated the involvement of the RNase III-like endoribonuclease, Bs-RNase III, in scRNA processing. Bs-RNase III was found to cleave precursor scRNA at two sites (the 5′ and 3′ cleavage sites) located on opposite sides of the stem of a large stem-loop structure, yielding a 275-nt RNA, which was then trimmed by a 3′ exoribonuclease to the mature scRNA. Here we show that Bs-RNase III cleaves primarily at the 5′ cleavage site and inefficiently at the 3′ site. RNase J1 is responsible for much of the cleavage that releases scRNA from downstream sequences. The subsequent exonucleolytic processing is carried out largely by RNase PH.     

RNase J is involved in the 5′-end maturation of 16S rRNA and 23S rRNA in Sinorhizobium meliloti

Sinorhizobium meliloti harbours genes encoding orthologs of ribonuclease (RNase) E and RNase J, the principle endoribonucleases in Escherichia coli and Bacillus subtilis, respectively. To analyse the role of RNase J in S. meliloti, RNA from a mutant with miniTn5-insertion in the RNase J-encoding gene was compared to the wild-type and a difference in the length of the 5.8S-like ribosomal RNA (rRNA) was observed. Complementation of the mutant, Northern blotting and primer extension revealed that RNase J is necessary for the 5′-end maturation of 16S rRNA and of the two 23S rRNA fragments, but not of 5S rRNA.     

Drosophila RNase Z processes mitochondrial and nuclear pre-tRNA 3' ends in vivo

Although correct tRNA 3′ ends are crucial for protein biosynthesis, generation of mature tRNA 3′ ends in eukaryotes is poorly understood and has so far only been investigated in vitro. We report here for the first time that eukaryotic tRNA 3′ end maturation is catalysed by the endonuclease RNase Z in vivo. Silencing of the JhI-1 gene (RNase Z homolog) in vivo with RNAi in Drosophila S2 cultured cells causes accumulation of nuclear and mitochondrial pre-tRNAs, suggesting that JhI-1 encodes both forms of the tRNA 3′ endonuclease RNase Z, and establishing its biological role in endonucleolytic tRNA 3′ end processing. In addition our data show that in vivo 5′ processing of nuclear and mitochondrial pre-tRNAs occurs before 3′ processing.     

Dissecting endonuclease and exonuclease activities in endonuclease V from Thermotoga maritima

Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3′-side 1 nt from a deaminated base lesion. DNA cleavage analysis using mutants defective in DNA binding and Mn²⁺ as a metal cofactor reveals a novel 3′-exonuclease activity in endonuclease V [Feng,H., Dong,L., Klutz,A.M., Aghaebrahim,N. and Cao,W. (2005) Defining amino acid residues involved in DNA-protein interactions and revelation of 3′-exonuclease activity in endonuclease V. Biochemistry, 44, 11486–11495.]. This study defines the enzymatic nature of the endonuclease and exonuclease activity in endonuclease V from Thermotoga maritima. In addition to its well-known inosine-dependent endonuclease, Tma endonuclease V also exhibits inosine-dependent 3′-exonuclease activity. The dependence on an inosine site and the exonuclease nature of the 3′-exonuclease activity was demonstrated using 5′-labeled and internally-labeled inosine-containing DNA and a H214D mutant that is defective in non-specific nuclease activity. Detailed kinetic analysis using 3′-labeled DNA indicates that Tma endonuclease V also possesses non-specific 5′-exonuclease activity. The multiplicity of the endonuclease and exonuclease activity is discussed with respect to deaminated base repair.     

Novel Protein-Protein Contacts Facilitate mRNA 3′-Processing Signal Recognition by Rna15 and Hrp1

Precise 3′-end processing of mRNA is essential for correct gene expression, yet in yeast, 3′-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3′-processing and are recognized by the RNA-binding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein-protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3′-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.     

3′-5′ tRNA guanylyltransferase in bacteria

The identity of the histidine specific transfer RNA (tRNA^His) is largely determined by a unique guanosine residue at position −1. In eukaryotes and archaea, the tRNA^His guanylyltransferase (Thg1) catalyzes 3′-5′ addition of G to the 5′-terminus of tRNA^His. Here, we show that Thg1 also occurs in bacteria. We demonstrate in vitro Thg1 activity for recombinant enzymes from the two bacteria Bacillus thuringiensis and Myxococcus xanthus and provide a closer investigation of several archaeal Thg1. The reaction mechanism of prokaryotic Thg1 differs from eukaryotic enzymes, as it does not require ATP. Complementation of a yeast thg1 knockout strain with bacterial Thg1 verified in vivo activity and suggests a relaxed recognition of the discriminator base in bacteria.     

Cell type-specific reciprocal regulation of HIF1A gene expression is dependent on 5′- and 3′-UTRs

In the present study, we demonstrated the reciprocal regulation of hypoxia-inducible factor 1 alpha (HIF1A) gene expression via untranslated region-(UTR) dependent mechanisms. A 151 nucleotide sequence found in the HIF1A 5′-UTR is sufficient for significant translational up-regulation. On the other hand, the 3′-UTR of HIF1A has been implicated in mRNA degradation. In the non-metastatic breast cancer cell line MCF7, the 3′-UTR-dependent down-regulatory machinery predominates over the 5′-UTR-dependent up-regulation of HIF1A. However, 5′-UTR-dependent up-regulation is dominant among metastatic cell lines (MDA-MB453, U87MG). It is therefore likely that the predominance of 5′-UTR-dependent translational enhancement of HIF1A is critical for the malignant phenotype of cancer cells. PTBP-1, but not HuR, is a candidate RNA binding protein for the translational control of HIF1A.     

The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome

Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3′ exonuclease activity. We report that expression only of Rrp44 lacking 3′-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5′-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3′-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endo–exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5′-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.