Distinct Contributions of Enzymic Functional Groups to the 2',3'-Cyclic Phosphodiesterase, 3'-Phosphate Guanylylation, and 3'-ppG/5'-OH Ligation Steps of the Escherichia coli RtcB Nucleic Acid Splicing Pathway.

UNLABELLED: Escherichia coli RtcB is a founding member of a family of manganese-dependent RNA repair enzymes that join RNA 2',3'-cyclic phosphate (RNA>p) or RNA 3'-phosphate (RNAp) ends to 5'-OH RNA (HORNA) ends in a multistep pathway whereby RtcB (i) hydrolyzes RNA>p to RNAp, (ii) transfers GMP from GTP to RNAp to form to RNAppG, and (iii) directs the attack of 5'-OH on RNAppG to form a 3'-5' phosphodiester splice junction. The crystal structure of the homologous archaeal RtcB enzyme revealed an active site with two closely spaced manganese ions, Mn1 and Mn2, that interact with the GTP phosphates. By studying the reactions of wild-type E. coli RtcB and RtcB alanine mutants with 3'-phosphate-, 2',3'-cyclic phosphate-, and 3'-ppG-terminated substrates, we found that enzymic constituents of the two metal coordination complexes (Cys78, His185, and His281 for Mn1 and Asp75, Cys78, and His168 for Mn2 in E. coli RtcB) play distinct catalytic roles. For example, whereas the C78A mutation abolished all steps assayed, the D75A mutation allowed cyclic phosphodiester hydrolysis but crippled 3'-phosphate guanylylation, and the H281A mutant was impaired in overall HORNAp and HORNA>p ligation but was able to seal a preguanylylated substrate. The archaeal counterpart of E. coli RtcB Arg189 coordinates a sulfate anion construed to mimic the position of an RNA phosphate. We propose that Arg189 coordinates a phosphodiester at the 5'-OH end, based on our findings that the R189A mutation slowed the step of RNAppG/HORNA sealing by a factor of 200 compared to that with wild-type RtcB while decreasing the rate of RNAppG formation by only 3-fold. IMPORTANCE: RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that ligate 2',3'-cyclic phosphate ends to 5'-OH ends via RNA 3'-phosphate and RNA(3')pp(5')G intermediates. The RtcB active site includes two adjacent manganese ions that engage the GTP phosphates. Alanine scanning of Escherichia coli RtcB reveals distinct contributions of metal-binding residues Cys78, Asp75, and His281 at different steps of the RtcB pathway. The RNA contacts of RtcB are uncharted. Mutagenesis implicates Arg189 in engaging the 5'-OH RNA end.

Characterization of 3'-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5'-Phosphate Capping Activity of RtcB3.

UNLABELLED: Escherichia coli RtcB exemplifies a family of GTP-dependent RNA repair/splicing enzymes that join 3'-PO4 ends to 5'-OH ends via stable RtcB-(histidinyl-N)-GMP and transient RNA3'pp5'G intermediates. E. coli RtcB also transfers GMP to a DNA 3'-PO4 end to form a stable "capped" product, DNA3'pp5'G. RtcB homologs are found in a multitude of bacterial proteomes, and many bacteria have genes encoding two or more RtcB paralogs; an extreme example is Myxococcus xanthus, which has six RtcBs. In this study, we purified, characterized, and compared the biochemical activities of three M. xanthus RtcB paralogs. We found that M. xanthus RtcB1 resembles E. coli RtcB in its ability to perform intra- and intermolecular sealing of a HORNAp substrate and capping of a DNA 3'-PO4 end. M. xanthus RtcB2 can splice HORNAp but has 5-fold-lower RNA ligase specific activity than RtcB1. In contrast, M. xanthus RtcB3 is distinctively feeble at ligating the HORNAp substrate, although it readily caps a DNA 3'-PO4 end. The novelty of M. xanthus RtcB3 is its capacity to cap DNA and RNA 5'-PO4 ends to form GppDNA and GppRNA products, respectively. As such, RtcB3 joins a growing list of enzymes (including RNA 3'-phosphate cyclase RtcA and thermophilic ATP-dependent RNA ligases) that can cap either end of a polynucleotide substrate. GppDNA formed by RtcB3 can be decapped to pDNA by the DNA repair enzyme aprataxin. IMPORTANCE: RtcB enzymes comprise a widely distributed family of RNA 3'-PO4 ligases distinguished by their formation of 3'-GMP-capped RNAppG and/or DNAppG polynucleotides. The mechanism and biochemical repertoire of E. coli RtcB are well studied, but it is unclear whether its properties apply to the many bacteria that have genes encoding multiple RtcB paralogs. A comparison of the biochemical activities of three M. xanthus paralogs, RtcB1, RtcB2, and RtcB3, shows that not all RtcBs are created equal. The standout findings concern RtcB3, which is (i) inactive as an RNA 3'-PO4 ligase but adept at capping a DNA 3'-PO4 end and (ii) able to cap DNA and RNA 5'-PO4 ends to form GppDNA and GppRNA, respectively. The GppDNA and GppRNA capping reactions are novel nucleic acid modifications.

Intact p53-dependent responses in miR-34-deficient mice.

MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34-deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation in vitro, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34-deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc-initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.