New Insights into the Mechanism of RNA Degradation by Ribonuclease II

RNase II is a key exoribonuclease involved in the maturation, turnover, and quality control of RNA. RNase II homologues are components of the exosome, a complex of exoribonucleases. The structure of RNase II unraveled crucial aspects of the mechanism of RNA degradation. Here we show that mutations in highly conserved residues at the active site affect the activity of the enzyme. Moreover, we have identified the residue that is responsible for setting the end product of RNase II. In addition, we present for the first time the models of two members of the RNase II family, RNase R from Escherichia coli and human Rrp44, also called Dis3. Our findings improve the present model for RNA degradation by the RNase II family of enzymes. RNase II is a key exoribonuclease involved in the maturation, turnover, and quality control of RNA. RNase II homologues are components of the exosome, a complex of exoribonucleases. The structure of RNase II unraveled crucial aspects of the mechanism of RNA degradation. Here we show that mutations in highly conserved residues at the active site affect the activity of the enzyme. Moreover, we have identified the residue that is responsible for setting the end product of RNase II. In addition, we present for the first time the models of two members of the RNase II family, RNase R from Escherichia coli and human Rrp44, also called Dis3. Our findings improve the present model for RNA degradation by the RNase II family of enzymes. Escherichia coli RNase II is the prototype of the RNase II superfamily of exoribonucleases, whose homologues are present in all three domains of life (1Mian I.S. Nucleic Acids Res. 1997; 25: 3187-3195Crossref PubMed Scopus (182) Google Scholar, 2Mitchell P. Petfalski E. Shevchenko A. Mann M. Tollervey D. Cell. 1997; 91: 457-466Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar, 3Grossman D. van Hoof A. Nat. Struct. Mol. Biol. 2006; 13: 760-761Crossref PubMed Scopus (11) Google Scholar, 4Zuo Y. Deutscher M.P. Nucleic Acids Res. 2001; 209: 1017-1026Crossref Scopus (395) Google Scholar). RNase II and its homologues can be environmentally (5Cairrão F. Chora A. Zilhão R. Carpousis J. Arraiano C.M. Mol. Microbiol. 2001; 276: 19172-19181Google Scholar, 6Andrade J.M. Cairrão F. Arraiano C.M. Mol. Microbiol. 2006; 60: 219-228Crossref PubMed Scopus (67) Google Scholar) and developmentally regulated (7Cairrão F. Arraiano C. Newbury S. Dev. Dyn. 2005; 232: 733-737Crossref PubMed Scopus (26) Google Scholar), and other ribonucleases have been shown to be involved in their post-transcriptional control (8Zilhão R. Cairrão F. Régnier P. Arraiano C.M. Mol. Microbiol. 1996; 20: 1033-1042Crossref PubMed Scopus (64) Google Scholar, 9Zilhão R. Regnier P. Arraiano C.M. FEMS Microbiol. Lett. 1995; 130: 237-244Crossref PubMed Google Scholar, 10Cairrão F. Arraiano C.M. Biochem. Biophys. Res. Commun. 2006; 343: 731-737Crossref PubMed Scopus (25) Google Scholar). Mutations in the rnb gene have been linked with abnormal chloroplast biogenesis (11Bollenbach T.J. Lange H. Gutierrez R. Erhardt M. Stern D.B. Gagliardi D. Nucleic Acids Res. 2005; 33: 2751-2763Crossref PubMed Scopus (91) Google Scholar), mitotic control, and cancer (12Lim J. Kuroki T. Ozaki K. Kohsaki H. Yamori T. Tsuruo T. Nakamori S. Imaoka S. Endo M. Nakamura Y. Cancer Res. 1997; 57: 921-925PubMed Google Scholar). RNase II is a ubiquitous enzyme that degrades single-stranded RNAs processively in the 3′-to5′-direction, resulting in the generation of 5′-mononucleotides. Ten nucleotides is the minimum length of the RNA molecule needed to detect activity in RNase II, and the end product of degradation of this enzyme is a 4-nucleotide RNA oligomer (13Amblar M. Barbas A. Fialho A.M. Arraiano C.M. J. Mol. Biol. 2006; 360: 921-933Crossref PubMed Scopus (64) Google Scholar, 14Cannistraro V.J. Kennell D. J. Mol. Biol. 1994; 243: 930-943Crossref PubMed Scopus (48) Google Scholar, 15Amblar M. Barbas A. Gomez-Puertas P. Arraiano C.M. RNA (N.Y.). 2007; 13: 317-327Crossref PubMed Scopus (44) Google Scholar). RNase II homologues Dis3/Rrp44 are components of the exosome, a complex of exoribonucleases involved in the maturation and turnover of RNA (2Mitchell P. Petfalski E. Shevchenko A. Mann M. Tollervey D. Cell. 1997; 91: 457-466Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar), in RNA interference (16Orban T.I. Izaurralde E. RNA (N.Y.). 2005; 11: 459-469Crossref PubMed Scopus (264) Google Scholar), and in surveillance pathways that recognize and degrade aberrant RNAs (17Lejeune F. Li X. Maquat L.E. Mol. Cell. 2003; 12: 675-687Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 18LaCava J. Houseley J. Saveanu C. Petfalski E. Thompson E. Jacquier A. Tollervey D. Cell. 2005; 121: 713-724Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). Recent reports have shown that Dis3/Rrp44 is the only catalytically active nuclease in the yeast core exosome (19Dziembowski A. Lorentzen E. Conti E. Seraphin B. Nat. Struct. Mol. Biol. 2007; 14: 15-22Crossref PubMed Scopus (329) Google Scholar) and plays a direct role in RNA surveillance, contributing to the recognition and degradation of specific RNA targets (20Schneider C. Anderson J.T. Tollervey D. Mol. Cell. 2007; 27: 324-331Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In addition, the human exosome has hydrolytic activity, similar to what happens in the yeast exosome (13Amblar M. Barbas A. Fialho A.M. Arraiano C.M. J. Mol. Biol. 2006; 360: 921-933Crossref PubMed Scopus (64) Google Scholar, 18LaCava J. Houseley J. Saveanu C. Petfalski E. Thompson E. Jacquier A. Tollervey D. Cell. 2005; 121: 713-724Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar, 21Liu Q. Greimann J.C. Lima C.D. Cell. 2006; 127: 1223-1237Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). 3C. D. Lima, personal communication. The RNase II homologues present in the exosome have the same behavior regarding the minimum length of RNA substrate and the same final end product as the E. coli RNase II (13Amblar M. Barbas A. Fialho A.M. Arraiano C.M. J. Mol. Biol. 2006; 360: 921-933Crossref PubMed Scopus (64) Google Scholar, 18LaCava J. Houseley J. Saveanu C. Petfalski E. Thompson E. Jacquier A. Tollervey D. Cell. 2005; 121: 713-724Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar, 21Liu Q. Greimann J.C. Lima C.D. Cell. 2006; 127: 1223-1237Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). Moreover, the recent determination of the electron microscopy structure of yeast Rrp44 (22Wang H.W. Wang J. Ding F. Callahan K. Bratkowski J. Butler J.S. Nogles E. Ke A. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 16844-16849Crossref PubMed Scopus (85) Google Scholar) showed that E. coli RNase II is a good model and suggests that the RNA recruitment mechanism is conserved. The three-dimensional structure of E. coli RNase II was recently determined (23Frazão C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar, 24McVey C.E. Amblar M. Barbas A. Cairrão F. Coelho R. Romão C. Arraiano C.M. Carrondo M.A. Frazão C. Acta Crystallogr. Sect. F. 2006; 62: 684-687Crossref PubMed Scopus (11) Google Scholar, 25Zuo Y. Vincent H.A. Zhang J. Wang Y. Deutscher M.P. Malhotra A. Mol. Cell. 2006; 24: 149-156Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The structure of RNase II RNA-bound complex (23Frazão C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar, 24McVey C.E. Amblar M. Barbas A. Cairrão F. Coelho R. Romão C. Arraiano C.M. Carrondo M.A. Frazão C. Acta Crystallogr. Sect. F. 2006; 62: 684-687Crossref PubMed Scopus (11) Google Scholar), together with biochemical data, gave new insights into the mechanisms of catalysis, translocation, and processivity of this important RNA-degrading enzyme. The E. coli RNase II structure and its RNA-bound complex have been used as a model for the analysis of Dis3/Rrp44 (20Schneider C. Anderson J.T. Tollervey D. Mol. Cell. 2007; 27: 324-331Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). RNase II consists of four domains (Fig. 1A): two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain. The RNA contacts the enzyme at two different and non-contiguous regions, the anchoring and the catalytic regions, which act synergistically to provide a processive degradation. Nucleotides 1 to 5 are located in the anchor region, situated in a deep cleft between the two cold shock domains and the S1 domain (Fig. 1A). The catalytic region forms a cavity that is only accessible to single-stranded RNA, where the last five nucleotides at the 3′-end of the RNA molecule (nt 9–13) are stacked and clamped between the aromatic residues Tyr-253 and Phe-358. In a recent report (23Frazão C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar) we postulated a model for RNA degradation that involves one Mg2+ ion, four highly conserved aspartic acids (201, 207, 209, and 210), and the recruitment of a second Mg2+ for catalysis. In this study we mutated these highly conserved amino acids present in the active site and characterized the respective proteins. Here, we also show, for the first time, the models of two related members of the RNase II family, RNase R from E. coli and human Rrp44 (Fig. 1, B and C). These results shed new light on the mechanism of RNA degradation by RNase II and can therefore be important for comprehension of the mode of action of other members of the RNase II family. Construction of RNase II Mutants by Site-directed Mutagenesis—Mutations D207N, D210N, F358A, and Y253A,F358A were introduced into pFCT6.9 (5Cairrão F. Chora A. Zilhão R. Carpousis J. Arraiano C.M. Mol. Microbiol. 2001; 276: 19172-19181Google Scholar) by PCR overlapping (26Higuchi, R. (1990) in PCR Protocols, A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) pp. 177-183 Academic Press, Inc./Harcourt Brace Jovanovich, San Diego, CAGoogle Scholar). D201N and Y253A mutations were generated by site-directed mutagenesis using QuikChange Site-directed Mutagenesis kit from Stratagene. The primers used in this study are described in supplemental experimental procedures. Overexpression and Purification of Wild-type and RNase II Mutants—The plasmid used for expression of wild-type E. coli histidine-tagged RNase II protein was pFCT6.9 plasmid (5Cairrão F. Chora A. Zilhão R. Carpousis J. Arraiano C.M. Mol. Microbiol. 2001; 276: 19172-19181Google Scholar). All other plasmids bearing mutations were transformed into BL21(DE3) E. coli strain (Novagen) to allow the expression of the recombinant proteins. Purification of all proteins was performed by histidine affinity chromatography using HiTrap Chelating HP columns (GE Healthcare) and the AKTA HLPC system (GE Healthcare) following the protocol previously described (13Amblar M. Barbas A. Fialho A.M. Arraiano C.M. J. Mol. Biol. 2006; 360: 921-933Crossref PubMed Scopus (64) Google Scholar). The purity of the enzymes was analyzed in an 8% SDS-PAGE (supplemental Fig. S1). Activity Assays—Exoribonucleolytic activity was assayed using oligoribonucleotides as substrate (13Amblar M. Barbas A. Fialho A.M. Arraiano C.M. J. Mol. Biol. 2006; 360: 921-933Crossref PubMed Scopus (64) Google Scholar). The 30-mer oligoribonucleotide (5′-CCCGACACCAACCACUAAAAAAAAAAAAAA-3′) and the poly(A) chain of 35 nt were labeled at the 5′-end with [γ-32P] ATP and T4 polynucleotide kinase. The RNA oligomers were then purified using Microcon YM-3 Centrifugal Filter Devices (Millipore). Reactions were carried out in a final volume of 10 μl containing 30 nm substrate, 20 mm Tris-HCl, pH 8, 100 mm KCl, 1 mm MgCl2, and 1 mm dithiothreitol. The amount of each enzyme added to the was to and is in Fig. Reactions were by the of the enzyme and at were at the time in the and the was by with 10 mm were in and analyzed by The activity of the enzymes was determined by and the of the substrate in three each the of these (supplemental Fig. The activity of the wild-type enzyme was as were from (GE The of the were with a of the as described in supplemental experimental procedures. The was at in a containing 20 mm Tris-HCl, pH 8, 100 mm KCl, 1 mm and mm The RNA substrate was on and the proteins were as in supplemental experimental procedures. All of each protein and to the of the and to control to the of the RNA the were using the to the model of RNase R and Rrp44 models for E. coli RNase R and human exosome complex Rrp44 proteins were performed using on the of the family as described in supplemental experimental procedures. of of Wild-type and Y253A a model of RNase the previously structure of RNase II with a poly(A) RNA (23Frazão C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar) was to of used was performed using the and the in the T. H. R. A. C. Wang B. J. 2005; PubMed Scopus Google Scholar, T. C. Wang J. R. Wang B. M. S. H. J. P. C. of San Scholar). on the in structure of the models for wild-type enzyme as as for Y253A were also generated by residues using procedures. were then to a second to the in the protein and its to the substrate RNA as a of the introduced on the are described in supplemental experimental procedures. to the RNase II, RNase and Rrp44 family of proteins were in protein using Zhang J. Zhang Nucleic Acids Res. 1997; 25: PubMed Scopus Google Scholar) and were using T.J. Nucleic Acids Res. 1994; PubMed Scopus Google Scholar) and C. J. J. Mol. Biol. PubMed Scopus Google Scholar) Mutations in the the RNA the the role of Tyr-253 and in RNA we mutated these aromatic residues into an and and In RNase II the RNA molecule (nt 9–13) is stacked and clamped between the aromatic residues Tyr-253 and Phe-358. on we that RNase II be in the of the of the RNA at the active site the of the substrate, catalysis. by Tyr-253 to of the activity of the enzyme 1 and Fig. models from the structure of the RNase complex in Tyr-253 stacked to the nt by its respective aromatic of and the to by the (Fig. and supplemental Fig. that Tyr-253 is important for the of the RNA in RNase II. its be other residues and contacts at the catalytic cavity that are for the RNA to to the In and located in to for a the model a of the protein these the was that the Y253A the product of degradation of RNase II from to 10 nt (Fig. an end product is that for a processive degradation by RNase II the RNA molecule to the anchoring and catalytic V.J. Kennell D. J. Mol. Biol. 1994; 243: 930-943Crossref PubMed Scopus (48) Google Scholar, C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar). Moreover, the three-dimensional model of the RNase complex that a is the minimum length of the RNA molecule that is to the anchoring and the catalytic (23Frazão C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar). RNA 10 nt only with the catalytic region, and degradation In Y253A the of Tyr-253 the of the RNA substrate at the catalytic site as a the at the anchoring region is for the RNA to to the enzyme. this RNA is to 10 nt and the RNA is then from the therefore the degradation product generated in the Y253A Tyr-253 an role in the of the 3′-end of the RNA substrate to the catalytic In the for Y253A by analysis is that of the wild-type enzyme 1 and supplemental Fig. and this that Y253A RNA Moreover, the of Y253A show the is the RNA a by of RNase II catalytic domain structure (Fig. and supplemental Fig. these to affect the of the residues in the active the catalytic activity of the Y253A activity and RNA affinity of wild-type and enzymes activity was assayed using a poly(A) chain as Activity were performed as described The were determined by using with a RNA oligomer activity of the wild-type enzyme was as The determination of the enzyme activity was carried out by and the of the activity of the wild-type enzyme was as The determination of the enzyme activity was carried out by and the of the substrate in a new the we the protein was active the wild-type the degradation product was similar to the was a product of 5 nt (Fig. that is as a to the last 5 nt the catalytic the final product is a the was by this was that of the wild-type that be a role in the of the RNA was by of the which to the by the three-dimensional structure suggests that the of the be in the in the T. H. R. A. C. Wang B. J. 2005; PubMed Scopus Google Scholar, T. C. Wang J. R. Wang B. M. S. H. J. P. C. of San Scholar). in with the experimental results for the a active role of this residue be with the is in to Tyr-253 the is present in all members of the RNase II family (supplemental Fig. models also that the role of these residues in RNA also be Tyr-253 be responsible for the of the at the 3′-end (Fig. the conserved the of the RNA by the to In by a as the RNA in that region, the of The Y253A,F358A similar to Y253A regarding the of the product the end product was nt (Fig. we a in activity the RNA affinity was that the the of Tyr-253 in RNA an of the RNA in the catalytic RNase II with other family we the models of E. coli RNase R and human Rrp44 proteins on the RNase II structure (Fig. 1, B and C). The results that these three enzymes a three-dimensional all the residues for activity located in the three protein models (Fig. is that have a of the amino In the of is to RNase II (supplemental Fig. RNase R protein a residue in the in with a similar in RNA an residue is in Rrp44 where is by residues with and (supplemental Fig. The in the amino in RNase II, RNase and Rrp44 enzymes their in to RNA degradation. the final end product of RNase II and Rrp44 is for RNase R is a (2Mitchell P. Petfalski E. Shevchenko A. Mann M. Tollervey D. Cell. 1997; 91: 457-466Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar, M. Barbas A. Fialho A.M. Arraiano C.M. J. Mol. Biol. 2006; 360: 921-933Crossref PubMed Scopus (64) Google Scholar, 15Amblar M. Barbas A. Gomez-Puertas P. Arraiano C.M. RNA (N.Y.). 2007; 13: 317-327Crossref PubMed Scopus (44) Google Scholar, C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar). The in RNase II in in highly conserved aspartic and are located in the RNase II active has been postulated that their is to the RNA substrate and the of the (23Frazão C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar, M. Arraiano C.M. J. 2005; PubMed Scopus Google Scholar). The of by in RNase II has been shown to be responsible for the of RNase II activity RNA M. Arraiano C.M. J. 2005; PubMed Scopus Google Scholar) 1 and Fig. A similar in the yeast RNase II Dis3/Rrp44 activity substrate and was responsible for a that the of this is important for yeast (19Dziembowski A. Lorentzen E. Conti E. Seraphin B. Nat. Struct. Mol. Biol. 2007; 14: 15-22Crossref PubMed Scopus (329) Google Scholar, C. Anderson J.T. Tollervey D. Mol. Cell. 2007; 27: 324-331Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). the role of these we mutated residues and to Our results that mutations in and to a of activity in degradation of poly(A) and of that of the wild-type and were to be the Mg2+ that is for together with two and two of the RNA substrate (23Frazão C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar). The of these by in the of one of the the at the active site and the in activity was with the 30 single-stranded substrate (Fig. D201N and generated a as a degradation in the as a product (Fig. The of these the of the RNA at the catalytic residues are contributing to the of the RNA at this region, the Mg2+ and (23Frazão C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar). 10 nt in the of the substrate to the anchor region is to a this the degradation of the 30 single-stranded to of a as a final product (Fig. substrate a poly(A) at its and D201N and enzymes degrade this to and then a by poly(A) substrate that has also been in the wild-type enzyme (13Amblar M. Barbas A. Fialho A.M. Arraiano C.M. J. Mol. Biol. 2006; 360: 921-933Crossref PubMed Scopus (64) Google Scholar, 14Cannistraro V.J. Kennell D. J. Mol. Biol. 1994; 243: 930-943Crossref PubMed Scopus (48) Google Scholar, 15Amblar M. Barbas A. Gomez-Puertas P. Arraiano C.M. RNA (N.Y.). 2007; 13: 317-327Crossref PubMed Scopus (44) Google Scholar) to be in these two The role of to be for with the other its by the enzyme activity 1 and Fig. from previously in which the of to to a of activity of that of the wild-type Y. Vincent H.A. Zhang J. Wang Y. Deutscher M.P. Malhotra A. Mol. Cell. 2006; 24: 149-156Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). the of an by other generated by the by in which only the of the residue is The structure of the RNase II that is of the of the RNA molecule (Fig. In this and was then in with the Mg2+ in the active its (Fig. the wild-type protein was and to was located in with the Mg2+ in the active resulting in the of to a (Fig. a of this to a only to the of the of and which in the activity of was in the degradation of the two 1 and Fig. the key role of in catalysis. In the three proteins D207N, and D210N, the between and only the wild-type RNase II enzyme only a in the RNA that 207, and are involved in the RNA In in the structure these three residues with the RNA substrate at the active site the Mg2+ by to the (23Frazão C. McVey C.E. Amblar M. Barbas A. Vonrhein C. Arraiano C.M. Carrondo M.A. Nature. 2006; 443: 110-114Crossref PubMed Scopus (189) Google Scholar). of by these mutations the of the RNA at the active Moreover, the three-dimensional models of E. coli RNase R and human Rrp44 proteins (Fig. 1, B and that these four aspartic residues have to in RNase II. we can similar catalytic for these residues in these two RNase enzymes. In this report we have the model for RNA degradation by RNase II by the role and of highly conserved residues present in the active site of the enzyme. that Tyr-253 and are for by RNase II, the enzyme activity the of these residues by Tyr-253 to be a residue in setting the product generated by RNase II the the of this residue in the of the 3′-end of the RNA Tyr-253 is highly conserved and residues are present in RNase II family members (supplemental Fig. into the and to Rrp44 (20Schneider C. Anderson J.T. Tollervey D. Mol. Cell. 2007; 27: 324-331Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), these be important for the of the role of this residue in and degradation of different RNA The of results in the by of the activity of the enzyme. suggests that be a degradation of the RNA by its translocation, to the of its aromatic between the of we show that the highly conserved and are and that their in RNA are with the only residue for RNase II Moreover, the models of E. coli RNase R and human Rrp44 proteins showed an structure similar to that of the RNase II, with the residues in similar In addition, the structure of yeast Rrp44 showed that E. coli RNase II is a good model and suggests that the RNA recruitment mechanism is conserved. we can and findings to related members of the RNase II family. study new light on the model previously for RNA degradation by RNase II. In addition, the that the RNase II is the only catalytically active nuclease in the exosome the to the role of highly conserved these results can be to the mechanisms for all RNase II family members that have a similar mode of present in the for and for on the analysis also for in and for with