Exoribonuclease R in Mycoplasma genitalium can carry out both RNA processing and degradative functions and is sensitive to RNA ribose methylation

Abstract

Mycoplasma genitalium, a small bacterium having minimal genome size, has only one identified exoribonuclease, RNase R (MgR). We have purified MgR to homogeneity, and compared its RNA degradative properties to those of its Escherichia coli homologs RNase R (EcR) and RNase II (EcII). MgR is active on a number of substrates including oligoribonucleotides, poly(A), rRNA, and precursors to tRNA. Unlike EcR, which degrades rRNA and pre-tRNA without formation of intermediate products, MgR appears sensitive to certain RNA structural features and forms specific products from these stable RNA substrates. The 39-ends of two MgR degradation products of 23S rRNA were mapped by RT-PCR to positions 2499 and 2553, each being 1 nucleotide downstream of a 29-O-methylation site. The sensitivity of MgR to ribose methylation is further demonstrated by the degradation patterns of 16S rRNA and a synthetic methylated oligoribonucleotide. Remarkably, MgR removes the 39-trailer sequence from a pre-tRNA, generating product with the mature 39-end more efficiently than EcII does. In contrast, EcR degrades this pre-tRNA without the formation of specific products. Our results suggest that MgR shares some properties of both EcR and EcII and can carry out a broad range of RNA processing and degradative functions.

Novel One-step Mechanism for tRNA 3′-End Maturation by the Exoribonuclease RNase R of Mycoplasma genitalium

“…RNA 5Ј-polyphosphatase was from Epicentre Biotechnologies (Madison, WI). The plasmid pET15b harboring the gene encoding M. genitalium RNase R (pETmgR) and the E. coli expression strain Rosetta-gami 2(DE3)pLysS were described previously (11). SequaGel for denaturing urea-PAGE was the product of National Diagnostics (Atlanta, GA).…”

“…Transformed cells were grown in LB medium at 37°C to A 600 ϭ 0.6, followed by induction using 1 mM isopropyl ␤-D-thiogalactopyranoside overnight at room temperature. Cells were harvested and processed, and M. genitalium RNase R was purified as described (11). Purified RNase R was at least 99% pure based on an SDS-PAGE analysis.…”

“…Surprisingly, none of the RNases listed above for tRNA 3Ј-maturation was identified in M. genitalium and related species (2,9,10). Recently, we demonstrated that purified RNase R of M. genitalium exhibits 3Ј 3 5Ј exoribonuclease activity that is somewhat different from the activities of its E. coli homologues RNases R and II (11). Interestingly, M. genitalium RNase R alone is able to remove 3Ј-trailers efficiently and to generate the mature tRNA 3Ј-end (11).…”

“…Interestingly, M. genitalium RNase R alone is able to remove 3Ј-trailers efficiently and to generate the mature tRNA 3Ј-end (11). In contrast, E. coli RNase R degrades pre-tRNA and other structural RNAs (11)(12)(13), whereas E. coli RNase II generates mature tRNA poorly (5,11). These observations suggest that in the presence of a limited number of RNases, M. genitalium RNase R may have acquired a unique exonucleolytic tRNA 3Ј-processing function.…”

“…Recently, we demonstrated that purified RNase R of M. genitalium exhibits 3Ј 3 5Ј exoribonuclease activity that is somewhat different from the activities of its E. coli homologues RNases R and II (11). Interestingly, M. genitalium RNase R alone is able to remove 3Ј-trailers efficiently and to generate the mature tRNA 3Ј-end (11). In contrast, E. coli RNase R degrades pre-tRNA and other structural RNAs (11)(12)(13), whereas E. coli RNase II generates mature tRNA poorly (5,11).…”