RNase D

Abstract: The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p displays a conserved RNase D core with a flanking HRDC (helicase and RNase D C-terminal) domain in an unusual conformation shown to be important for the processing function of the enzyme. Complexes with AMP and UMP, the products of the RNA degradation process, reveal how the protein specifically recognizes ribonucleotides and their bases. Finally, in vivo mutational studies show the importance of the domain contacts for the processing function of Rrp6p and highlight fundamental differences between the protein and its prokaryotic RNase D counterparts.

Abstract: Our knowledge of the 3' processing of tRNA precursors is severely limited. Although six exoribonucleases able to act on Escherichia coli tRNA precursors in vitro have been identified, their involvement in tRNA maturation in vivo has not been demonstrated. Here we show, using a wide range of multiple RNase-deficient strains and a quantitative suppression assay, that at least five of these enzymes--RNase II, RNase D, RNase BN, RNase T, and RNase PH--can participate in the synthesis of functional tRNA(Tyr)su+3 in vivo. Moreover, any one of the five RNases is sufficient to allow tRNA processing to proceed although with varying effectiveness. Examination of the level of aminoacylation of tRNA isolated from RNase-deficient strains suggested that tRNA precursors accumulate in the most defective cells. These data indicate that exoribonucleases are required for tRNA maturation in vivo and that there is a high degree of functional overlap among the enzymes. These studies contribute to the identification of all the ...

Abstract: The 3′–5′ riboexonuclease Rrp6p, a nuclear component of the exosome, functions with other exosome components to produce the mature 3′ ends of 5.8S rRNA, sno- and snRNAs, and to destroy improperly processed precursor (pre)-rRNAs and pre-mRNAs. Rrp6p is a member of the RNase D family of riboexonucleases and displays a high degree of homology with the active site of the deoxyriboexonuclease domain of Escherichia coli DNA polymerase I, the crystal structure of which indicates a two-metal ion mechanism for phosphodiester bond hydrolysis. Mutation of each of the conserved residues predicted to coordinate metal ions in the active site of Rrp6p abolished activity of the enzyme in vitro and in vivo. Complete loss of Rrp6p activity caused by the Y361F and Y361A mutations supports the critical role proposed for the phenolic hydroxyl of Tyr361 in the reaction mechanism. Rrp6p also contains an helicase RNase D C-terminal (HRDC) domain of unknown function that is similar to domains in the Werner’s and Bloom’s Syndrome proteins. A point mutation in this domain results in Rrp6p that localizes to the nucleus, but fails to efficiently process the 3′ ends of 5.8S pre-rRNA and some pre-snoRNAs. In contrast, this mutant retains the ability to degrade rRNA processing intermediates and 3′-extended, poly(A)+ snoRNAs. These findings indicate the potential for independent control of the processing and degradation functions of Rrp6p.

Abstract: Examination of double mutants lacking one of the exoribonucleases, RNase II, RNase D, RNase BN, or RNase R, and also devoid of tRNA nucleotidyltransferase has suggested that none of these RNases participates in the end-turnover of tRNA. This prompted a search for and identification of a new exoribonuclease, termed RNase T. RNase T could be detected in mutant Escherichia coli strains lacking as many as three of the known exoribonucleases, and it could be separated from each of the four previously described RNases. RNase T is optimally active at pH 8-9 and requires a divalent cation for activity. The enzyme is sensitive to ionic strengths greater than 50 mM and is rapidly inactivated by heating at 45 degrees C. Its preferred substrate is tRNA-C-C-[14C]A, with much less activity shown against tRNA-C-C. RNase T is an exoribonuclease that initiates attack at the 3' hydroxyl terminus of tRNA and releases AMP in a random mode of hydrolysis. The possible involvement of RNase T in end-turnover of tRNA and in RNA metabolism in general are discussed.