RNA Editing Associated with the Generation of Two Distinct Conformations of the Trypanosomatid Leptomonas collosoma 7SL RNA
1999; Elsevier BV; Volume: 274; Issue: 36 Linguagem: Inglês
10.1074/jbc.274.36.25642
ISSN1083-351X
AutoresHerzel Ben-Shlomo, Alexander Levitan, Naomi Editha Shay, И. Н. Гончаров, Shulamit Michaeli,
Tópico(s)Research on Leishmaniasis Studies
ResumoAnalysis of the trypanosomatid Leptomonas collosoma 7SL RNA revealed the existence of two distinct stable 7SL RNA conformers (7SL I and II). Sequence analysis of the RNAs indicated a single base difference between the conformers at position 133 (C in 7SL II and U in 7SL I) located in domain III. This change appears to be the result of a post-transcriptional editing event, since the single-copy 7SL RNA gene codes exclusively for a C at this position. The edited form (7SL I) was found preferentially in the cytoplasm, and the pre-edited form in the nucleus. 7SL I is mainly bound to ribosomes, whereas 7SL II is more abundant in ribosome-free particles. Mutations introduced in regions outside the editing site were found to occur in a single conformation, suggesting that the editing event is not the only factor that determines the conformation of the molecule. This study is the first description of an editing event on a small RNA other than tRNA and is the first report of C → U editing in trypanosomes. We propose a novel role for RNA editing in controlling the conformation of the 7SL RNA in vivo. Analysis of the trypanosomatid Leptomonas collosoma 7SL RNA revealed the existence of two distinct stable 7SL RNA conformers (7SL I and II). Sequence analysis of the RNAs indicated a single base difference between the conformers at position 133 (C in 7SL II and U in 7SL I) located in domain III. This change appears to be the result of a post-transcriptional editing event, since the single-copy 7SL RNA gene codes exclusively for a C at this position. The edited form (7SL I) was found preferentially in the cytoplasm, and the pre-edited form in the nucleus. 7SL I is mainly bound to ribosomes, whereas 7SL II is more abundant in ribosome-free particles. Mutations introduced in regions outside the editing site were found to occur in a single conformation, suggesting that the editing event is not the only factor that determines the conformation of the molecule. This study is the first description of an editing event on a small RNA other than tRNA and is the first report of C → U editing in trypanosomes. We propose a novel role for RNA editing in controlling the conformation of the 7SL RNA in vivo. signal recognition particle endoplasmic reticulum ribonucleoprotein nucleotide(s) post-ribosomal supernatant avian myeloblastosis virus diethyl pyrocarbonate spliced leader small nucleolar RNA The signal recognition particle (SRP)1 functions as an adaptor between the protein synthesis machinery and the protein translocation apparatus (1Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (713) Google Scholar). SRP was shown in vitro to bind the signal sequence emerging from translating ribosomes and to trigger a transient pause in elongation. The arrest in translation was documented by canine SRP in a wheat-germ cell-free system (2Walter P. Blobel G. J. Cell Biol. 1981; 91: 557-561Crossref PubMed Scopus (456) Google Scholar) but not when canine SRP was added to either reticulocyte lysate or HeLa cell extracts (3Meyer D.I. EMBO J. 1985; 4: 2031-2033Crossref PubMed Scopus (32) Google Scholar). However, SRP was shown to cause translational pausing at multiple sites in the nascent polypeptide in reticulocyte lysates (4Wolin S.L. Walter P. J. Cell Biol. 1993; 121: 1211-1219Crossref PubMed Scopus (39) Google Scholar). In the second step of the SRP cycle, elongation arrest is relieved when SRP interacts with the SRP receptor. The SRP is released and can recycle, whereas the ribosome remains attached to the membrane and the nascent chain is then translocated co-translationally into the lumen of the endoplasmic reticulum (ER).The best studied eukaryotic SRP is the canine particle that is composed of one RNA molecule, the 7SL RNA, and six proteins: SRPs 9, 14, 19, 54, 68, and 72 (5Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (460) Google Scholar). In vitro studies with canine SRP indicated that SRP54 binds the signal peptide as it emerges from the ribosome, SRP9/14 bind to domain I and function in elongation arrest, SRP68/72 promote translocation into the ER, whereas SRP19 facilitates the binding of SRP54 to the RNA (6Siegel V. Walter P. Trends Biochem. Sci. 1988; 3: 314-316Abstract Full Text PDF Scopus (43) Google Scholar). SRP exists in the cell in different states. About 15% of the SRPs are found as free (∼11 S) particles, and the remainder are divided almost equally between ribosomes and microsomal membranes (7Walter P. Blobel G. J. Cell Biol. 1983; 97: 1693-1699Crossref PubMed Scopus (44) Google Scholar). The association of SRPs to monosomes is weak compared with its tight binding to polysomes (7Walter P. Blobel G. J. Cell Biol. 1983; 97: 1693-1699Crossref PubMed Scopus (44) Google Scholar).7SL RNA was cloned and sequenced from a variety of eukaryotes and these RNAs appear to fit a canonical secondary structure model (8Althoff S. Selinger D. Wise J.A. Nucleic Acids Res. 1994; 22: 1933-1947Crossref PubMed Scopus (74) Google Scholar). Despite extensive phylogenetic studies on the 7SL RNA, the only experimental data supporting the secondary structure model are the nuclease digestion (9Gundelfinger E.D. Carlo M.D. Zopf D. Melli M. EMBO J. 1984; 3: 2325-2332Crossref PubMed Scopus (56) Google Scholar) and α-sarcin cleavage data (10Siegel V. Walter P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1801-1805Crossref PubMed Scopus (84) Google Scholar). The trypanosome 7SL RNA also fits the canonical secondary structure model, except that domain I appear to deviate in length and structure from the human RNA (11Michaeli S. Podell D. Agabian N. Ullu E. Mol. Biochem. Parasitol. 1992; 1: 55-65Crossref Scopus (33) Google Scholar, 12Béjà O. Ullu E. Michaeli S. Mol. Biochem. Parasitol. 1993; 57: 223-230Crossref PubMed Scopus (30) Google Scholar, 13Ben-Shlomo H. Levitan A. Béjà O. Michaeli S. Nucleic Acids Res. 1997; 25: 4977-4984Crossref PubMed Scopus (22) Google Scholar). Only 70% identity exists between the 7SL RNAs of the monogenetic trypanosomatid Leptomonas collosoma and Trypanosoma brucei (13Ben-Shlomo H. Levitan A. Béjà O. Michaeli S. Nucleic Acids Res. 1997; 25: 4977-4984Crossref PubMed Scopus (22) Google Scholar). Whereas domains I and IV of these trypanosomatid 7SL RNAs are highly conserved, domain III is divergent (13Ben-Shlomo H. Levitan A. Béjà O. Michaeli S. Nucleic Acids Res. 1997; 25: 4977-4984Crossref PubMed Scopus (22) Google Scholar). The presence of a co-migrating tRNA-like molecule, which co-purifies with the T. brucei 7SL RNA (12Béjà O. Ullu E. Michaeli S. Mol. Biochem. Parasitol. 1993; 57: 223-230Crossref PubMed Scopus (30) Google Scholar), led us to hypothesize that the trypanosomatid SRP may differ from other SRPs and is composed of two small RNAs. Using affinity selection with antisense biotinylated oligonucleotide, we have recently demonstrated that L. collosoma SRP complex is indeed composed of two RNA molecules, the 7SL RNA and a tRNA-like molecule (sRNA-85). 2H. Ben-Shlomo, Y.-X. Xu, L. Liu, Y. Zhang, I. Goncharov, and S. Michaeli, submitted for publication.2H. Ben-Shlomo, Y.-X. Xu, L. Liu, Y. Zhang, I. Goncharov, and S. Michaeli, submitted for publication.The exact function of the 7SL RNA within the SRP complex is unknown. 7SL RNA most probably does not function simply as a passive scaffold for the SRP proteins but rather has an active role in the translocation process. SRP may therefore undergo structural rearrangements during the functional cycle of the SRP. Indeed, one study, performed on soluble polysome-bound and membrane-bound SRPs, revealed that the secondary structure of the 7SL RNA in these particles is different, suggesting that the 7SL RNA may play an active role in the SRP cycle (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar). The potential for the 7SL RNA to exist in more than one conformation was observed for human 7SL RNA (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar, 15Zwieb C. Ullu E. Nucleic Acids Res. 1986; 4: 4639-4658Crossref Scopus (24) Google Scholar, 16Gowda K. Zwieb C. Nucleic Acids Res. 1997; 25: 2835-2840Crossref PubMed Scopus (18) Google Scholar) and the sequences that are essential for adopting the different conformations were determined (15Zwieb C. Ullu E. Nucleic Acids Res. 1986; 4: 4639-4658Crossref Scopus (24) Google Scholar,16Gowda K. Zwieb C. Nucleic Acids Res. 1997; 25: 2835-2840Crossref PubMed Scopus (18) Google Scholar).RNA editing is a process that co or post-transcriptionally modifies RNA primary sequence from that encoded by the gene by either deletion, insertion, or base modification (17Smith H.C. Gott J.A. Hanson M.R. RNA. 1997; 3: 1105-1123PubMed Google Scholar). The phenomenon was first described in trypanosomatid mitochondrial mRNA and was shown to occur through a guide RNA-mediated cleavage and ligation (18Stuart K. Allen T.E. Kable M.L. Lawson S. Curr. Opin. Chem. Biol. 1997; 1: 340-346Crossref PubMed Scopus (15) Google Scholar). RNA editing has since been found in diverse lower and higher eukaryotes. The process can occur in the nucleus, chloroplast, and mitochondria, and it involves base modification such as C → U, U → C, and A → I transitions on mRNAs (17Smith H.C. Gott J.A. Hanson M.R. RNA. 1997; 3: 1105-1123PubMed Google Scholar). RNA editing was also shown to alter the sequence of tRNA and rRNA (17Smith H.C. Gott J.A. Hanson M.R. RNA. 1997; 3: 1105-1123PubMed Google Scholar).In this study, we demonstrate that the L. collosoma 7SL RNA is present in the cell in two stable conformational states. The conformational change is associated with an RNA editing event of C → U conversion at position 133. This event, however, is not the only factor that determines whether 7SL RNA will undergo the structural changes, since 7SL RNA mutants, altered in regions outside the editing site, were found in a single conformation. The conversion between the two conformations is a dynamic process that takes place in the cytoplasm; 7SL I was also found preferentially attached to ribosomes. This is the first report that correlates conformational change of a small RNA molecule with RNA editing and is the first to report the existence of C → U editing in trypanosomes.DISCUSSIONThis study demonstrates that the trypanosomatid 7SL RNA undergoes a conformational change in vivo that is associated with an RNA editing event. This is the first description of a C → U editing on a small RNA apart from tRNA, and is also the first case of C → U editing in trypanosomes.The L. collosoma 7SL RNA, unlike all 7SL RNA described so far, is not fully denatured in 7 m urea. Such a property was previously observed for E. coli 5 S rRNA (26Digweed M. Kumagai I. Pieler T. Erdmann V.A. Eur. J. Biochem. 1982; 127: 531-537Crossref PubMed Scopus (12) Google Scholar). In the latter case, the fast migrating molecule carried a U in position 92, whereas the slow migrating variant contained a C in the same position (26Digweed M. Kumagai I. Pieler T. Erdmann V.A. Eur. J. Biochem. 1982; 127: 531-537Crossref PubMed Scopus (12) Google Scholar). This is exactly the case in this study, since the fast migrating 7SL I carries a U in position 133 and the slow migrating molecule (7SL II) carries a C in the same position. In the case of the 5 S rRNA, it was suggested that the only difference observed between the two variants is responsible for the drastic reduction in the stability of the two 5 S rRNA molecules (26Digweed M. Kumagai I. Pieler T. Erdmann V.A. Eur. J. Biochem. 1982; 127: 531-537Crossref PubMed Scopus (12) Google Scholar). It is currently unknown whether this is also the case in the L. collosoma 7SL RNA. However, the findings that mutated 7SL RNAs located outside the editing site were found in a single conformation may suggest that the editing is not the only factor that controls the structural change. The mechanism that elicits the conformational change of 7SL RNA is unknown. Since the editing site is situated close to loop III and to the region that was shown to bind SRP19 in the canine SRP (30Siegel V. Walter P. Proc. Natl. Acad. Sci U. S. A. 1988; 85: 1801-1805Crossref PubMed Scopus (86) Google Scholar), it may alter SRP19 binding. Alternatively, the editing event may affect long range tertiary interactions.The differences in pause sites observed between 7SL I and 7SL II may represent regions on the molecule that present obstacles for reverse transcriptase due to RNA structures that are not melted because of strong secondary or tertiary interactions. The differential stops between 7SL I and 7SL II reflect differences in the structures of the two molecules. These different structures are mostly located in domains II and III. Previous studies performed on naked human 7SL RNA indicated that in vitro transcribed human 7SL RNA can exist in two different conformations that can be separated on native gels (15Zwieb C. Ullu E. Nucleic Acids Res. 1986; 4: 4639-4658Crossref Scopus (24) Google Scholar, 16Gowda K. Zwieb C. Nucleic Acids Res. 1997; 25: 2835-2840Crossref PubMed Scopus (18) Google Scholar). By site-directed mutagenesis, different sites were shown to be important for the formation of these two conformations. Most of the mutations affecting the conformation of the RNA were located in domains II and III of the RNA (16Gowda K. Zwieb C. Nucleic Acids Res. 1997; 25: 2835-2840Crossref PubMed Scopus (18) Google Scholar). These are also the domains where major differences were seen in stop sites between the trypanosomatid 7SL I and II. A small region located between positions 129 to 134 of the mammalian 7SL RNA, which is part of the SRP 19 binding site was shown to be most critical for the conformational change that the mammalian 7SL RNA undergoes (16Gowda K. Zwieb C. Nucleic Acids Res. 1997; 25: 2835-2840Crossref PubMed Scopus (18) Google Scholar). The editing site detected in this study is located in this same domain.Studies performed on ribosome-bound, membrane-bound, and free canine SRPs have indicated that the 7SL RNAs in these SRPs are found in different conformations. This conclusion was based on variation in sensitivity of the different SRPs to chemical modifications (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar). Initially, it was expected that polysome-bound SRP should be less sensitive to chemical modification than free SRPs because of potential protection of the particles by ribosomes. Instead, the converse was found, and chemical accessibility of polysome-bound SRP was actually higher than that of soluble SRPs, suggesting that the polysome-bound SRP has a more open conformation than that of the 7SL RNA in free particles (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar). The results presented in this study supports this notion, since 7SL I, which is preferentially associated with ribosomes, is the molecule that was found to be more accessible to chemical modification and therefore has a more open structure, whereas the 7SL II, which is found mostly associated with free SRPs possesses a more closed structure, as revealed by the many strong pause sites and inaccessibility to modification by DEPC. The location of the pause sites observed on the 7SL RNA agrees well with the regions that were shown to be involved in binding to ribosomes or to the rough ER membrane (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar). In particular, attention should be drawn to the region around nt 120 in domain III, since homologous regions on the mammalian 7SL RNA were shown to bind to ribosomes or the ER membrane. In addition, the regions in domain II around nt 220 were also shown in the mammalian system to be involved in binding to ribosomes or ER membrane (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar). Interestingly, only domain I of the 7SL I but not of 7SL II was accessible to interaction with DEPC, suggesting major differences in the structure of this Alu-like domain between the conformers. In the mammalian SRP, however, the Alu domain was less accessible to chemical modification compared with S domains II and III (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar).It is currently unknown why the 7SL RNA undergoes a conformational change during the translocation cycle. Two steps in the translocation cycle may require changes in the 7SL RNA: first when SRP interacts with the ribosome and induces an arrest or a pause in translation, and again when SRP is released from the ribosome after its interaction with the SRP receptor (1Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (713) Google Scholar). Conformational changes of rRNA have been shown to take place in the transition from inactive to active 30 S ribosomal subunits (31Moazed D. Noller H.F. Cell. 1986; 47: 985-994Abstract Full Text PDF PubMed Scopus (350) Google Scholar), in the assembly of subunits to monosomes (32Stebbins-Boaz B. Gerbi S.A. J. Mol. Biol. 1990; 217: 93-112Crossref Scopus (15) Google Scholar) and during tRNA translocation (33Moazed D. Noller H.F. Nature. 1989; 342: 142-148Crossref PubMed Scopus (606) Google Scholar). In this context, the 7SL RNA may cause translation arrest by interacting with the rRNA, thus interfering with the conformational changes rRNA undergoes during protein synthesis. However, the arrest function of SRP was shown to be mediated by domain I (6Siegel V. Walter P. Trends Biochem. Sci. 1988; 3: 314-316Abstract Full Text PDF Scopus (43) Google Scholar). It has been suggested that domain I functions in protein arrest by mimicking the shape of a tRNA and thereby blocking the entry of incoming tRNAs. Our finding that a tRNA-like molecule is present in the SRP complex in a 1:1 ratio with the 7SL RNA supports the notion that the tRNA-like domain may play a role in the arrest (2Walter P. Blobel G. J. Cell Biol. 1981; 91: 557-561Crossref PubMed Scopus (456) Google Scholar). The data presented in this paper, as well as the studies on the canine SRP (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar) demonstrating that the structure of domains II and III are different in ribosome-free and ribosome-bound SRP, suggest that additional interactions apart from the domain I but with domains II and III of the molecule are also essential for the interaction of the SRP with the ribosome. Further studies are needed to accurately map the site of interaction of 7SL RNA with rRNA, e.g. by in vivo UV-induced psoralen cross-linking.C → U conversion is among the best documented RNA editing events and has been shown to take place in mitochondria, chloroplast, and the nucleus (17Smith H.C. Gott J.A. Hanson M.R. RNA. 1997; 3: 1105-1123PubMed Google Scholar). The first reported C → U editing by base deamination was for the apolipoprotein B mRNA that takes place in the nucleus post-transcriptionally (17Smith H.C. Gott J.A. Hanson M.R. RNA. 1997; 3: 1105-1123PubMed Google Scholar). C → U editing was found also in the mitochondria and chloroplast of land plants (34Marchfelder A. Binder S. Brennicke A. Knoop V. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 307-323Google Scholar). This type of editing is, however, not restricted to mRNA and was found to modify the anticodon loop and acceptor stem of tRNAs (35Price D. Gray M.W. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 289-305Google Scholar). Whereas the editing of apoB mRNA is directed by a mooring sequence located adjacent to the editing site, tRNA editing, especially editing sites in the acceptor stem, may be guided by the base-paired region opposite the edited nucleotide (34Marchfelder A. Binder S. Brennicke A. Knoop V. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 307-323Google Scholar, 35Price D. Gray M.W. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 289-305Google Scholar). This could also be the case for the 7SL RNA editing, since the site is present in an 8-base pair stem-loop structure. It is currently unknown what type of activity could mediate the editing event revealed in this study. However, the conversion of C → U may "freeze" the 7SL I. Since the results presented in this study suggest that the conversion of 7SL I to 7SL II is a dynamic process, it may imply that an activity that mediates U → C conversion should also exist to convert 7SL I to 7SL II. U → C editing was reported mainly in the mitochondria and chloroplast of land plants and mammalian tRNAs (34Marchfelder A. Binder S. Brennicke A. Knoop V. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 307-323Google Scholar). This conversion could be achieved by transamination.The finding of C → U editing in trypanosomes is especially interesting because it suggests that U insertion and deletion present in the kinetoplast (18Stuart K. Allen T.E. Kable M.L. Lawson S. Curr. Opin. Chem. Biol. 1997; 1: 340-346Crossref PubMed Scopus (15) Google Scholar) is not the only editing pathway in trypanosomes and that two different mechanisms of editing can co-exist in the same organism. Because of the early divergence of trypanosomes from the eukaryotic lineage, the finding of C → U editing in trypanosomes suggests that this process evolved early in eukaryotic evolution.The localization of 7SL I in the cytoplasmic fraction suggests that this editing takes place in the cytoplasm. We could not, however, rule out the possibility that editing takes place in the nucleus and that the edited RNA is rapidly translocated to the cytoplasm. The finding that 7SL RNA mutants examined in this study exist in a single conformation may suggest that only 7SL RNA molecules that engage active particles undergo the conformational change. We therefore favor the hypothesis that the editing function may be associated with the ribosome. This hypothesis is not unprecedented, since deaminases involved in editing were found in the cytoplasm of plants (34Marchfelder A. Binder S. Brennicke A. Knoop V. Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998: 307-323Google Scholar).The combination of the unique properties of the L. collosoma 7SL RNA with the ability to express in vivo mutated 7SL RNA will be further used to identify sequences that are involved in: (a) the editing event, (b) the ability to undergo the conformational change, and (c) binding of ribosomes. The establishment of an in vitro system that is amenable to convert 7SL II is essential for better understanding the mechanism and machinery that carries out this novel editing event. We anticipate that other editing events of C → U on small RNAs, such as tRNAs and other cellular and kinetoplast mRNAs, may be found in trypanosomes. The signal recognition particle (SRP)1 functions as an adaptor between the protein synthesis machinery and the protein translocation apparatus (1Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (713) Google Scholar). SRP was shown in vitro to bind the signal sequence emerging from translating ribosomes and to trigger a transient pause in elongation. The arrest in translation was documented by canine SRP in a wheat-germ cell-free system (2Walter P. Blobel G. J. Cell Biol. 1981; 91: 557-561Crossref PubMed Scopus (456) Google Scholar) but not when canine SRP was added to either reticulocyte lysate or HeLa cell extracts (3Meyer D.I. EMBO J. 1985; 4: 2031-2033Crossref PubMed Scopus (32) Google Scholar). However, SRP was shown to cause translational pausing at multiple sites in the nascent polypeptide in reticulocyte lysates (4Wolin S.L. Walter P. J. Cell Biol. 1993; 121: 1211-1219Crossref PubMed Scopus (39) Google Scholar). In the second step of the SRP cycle, elongation arrest is relieved when SRP interacts with the SRP receptor. The SRP is released and can recycle, whereas the ribosome remains attached to the membrane and the nascent chain is then translocated co-translationally into the lumen of the endoplasmic reticulum (ER). The best studied eukaryotic SRP is the canine particle that is composed of one RNA molecule, the 7SL RNA, and six proteins: SRPs 9, 14, 19, 54, 68, and 72 (5Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (460) Google Scholar). In vitro studies with canine SRP indicated that SRP54 binds the signal peptide as it emerges from the ribosome, SRP9/14 bind to domain I and function in elongation arrest, SRP68/72 promote translocation into the ER, whereas SRP19 facilitates the binding of SRP54 to the RNA (6Siegel V. Walter P. Trends Biochem. Sci. 1988; 3: 314-316Abstract Full Text PDF Scopus (43) Google Scholar). SRP exists in the cell in different states. About 15% of the SRPs are found as free (∼11 S) particles, and the remainder are divided almost equally between ribosomes and microsomal membranes (7Walter P. Blobel G. J. Cell Biol. 1983; 97: 1693-1699Crossref PubMed Scopus (44) Google Scholar). The association of SRPs to monosomes is weak compared with its tight binding to polysomes (7Walter P. Blobel G. J. Cell Biol. 1983; 97: 1693-1699Crossref PubMed Scopus (44) Google Scholar). 7SL RNA was cloned and sequenced from a variety of eukaryotes and these RNAs appear to fit a canonical secondary structure model (8Althoff S. Selinger D. Wise J.A. Nucleic Acids Res. 1994; 22: 1933-1947Crossref PubMed Scopus (74) Google Scholar). Despite extensive phylogenetic studies on the 7SL RNA, the only experimental data supporting the secondary structure model are the nuclease digestion (9Gundelfinger E.D. Carlo M.D. Zopf D. Melli M. EMBO J. 1984; 3: 2325-2332Crossref PubMed Scopus (56) Google Scholar) and α-sarcin cleavage data (10Siegel V. Walter P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1801-1805Crossref PubMed Scopus (84) Google Scholar). The trypanosome 7SL RNA also fits the canonical secondary structure model, except that domain I appear to deviate in length and structure from the human RNA (11Michaeli S. Podell D. Agabian N. Ullu E. Mol. Biochem. Parasitol. 1992; 1: 55-65Crossref Scopus (33) Google Scholar, 12Béjà O. Ullu E. Michaeli S. Mol. Biochem. Parasitol. 1993; 57: 223-230Crossref PubMed Scopus (30) Google Scholar, 13Ben-Shlomo H. Levitan A. Béjà O. Michaeli S. Nucleic Acids Res. 1997; 25: 4977-4984Crossref PubMed Scopus (22) Google Scholar). Only 70% identity exists between the 7SL RNAs of the monogenetic trypanosomatid Leptomonas collosoma and Trypanosoma brucei (13Ben-Shlomo H. Levitan A. Béjà O. Michaeli S. Nucleic Acids Res. 1997; 25: 4977-4984Crossref PubMed Scopus (22) Google Scholar). Whereas domains I and IV of these trypanosomatid 7SL RNAs are highly conserved, domain III is divergent (13Ben-Shlomo H. Levitan A. Béjà O. Michaeli S. Nucleic Acids Res. 1997; 25: 4977-4984Crossref PubMed Scopus (22) Google Scholar). The presence of a co-migrating tRNA-like molecule, which co-purifies with the T. brucei 7SL RNA (12Béjà O. Ullu E. Michaeli S. Mol. Biochem. Parasitol. 1993; 57: 223-230Crossref PubMed Scopus (30) Google Scholar), led us to hypothesize that the trypanosomatid SRP may differ from other SRPs and is composed of two small RNAs. Using affinity selection with antisense biotinylated oligonucleotide, we have recently demonstrated that L. collosoma SRP complex is indeed composed of two RNA molecules, the 7SL RNA and a tRNA-like molecule (sRNA-85). 2H. Ben-Shlomo, Y.-X. Xu, L. Liu, Y. Zhang, I. Goncharov, and S. Michaeli, submitted for publication.2H. Ben-Shlomo, Y.-X. Xu, L. Liu, Y. Zhang, I. Goncharov, and S. Michaeli, submitted for publication. The exact function of the 7SL RNA within the SRP complex is unknown. 7SL RNA most probably does not function simply as a passive scaffold for the SRP proteins but rather has an active role in the translocation process. SRP may therefore undergo structural rearrangements during the functional cycle of the SRP. Indeed, one study, performed on soluble polysome-bound and membrane-bound SRPs, revealed that the secondary structure of the 7SL RNA in these particles is different, suggesting that the 7SL RNA may play an active role in the SRP cycle (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar). The potential for the 7SL RNA to exist in more than one conformation was observed for human 7SL RNA (14Andreazzoli M. Gerbi S.A. EMBO J. 1991; 10: 767-777Crossref PubMed Scopus (42) Google Scholar, 15Zwieb C. Ullu E. Nucleic Acids Res. 1986; 4: 4639-4658Crossref Scopus (24) Google Scholar, 16Gowda K. Zwieb C. Nucleic Acids Res. 1997; 25: 2835-2840Crossref PubMed Scopus (18) Google Scholar) and the sequences that are essential for adopting the different conformations were determined (15Zwieb C. Ullu E. Nucleic Acids Res. 1986; 4: 4639-4658Crossref Scopus (24) Google Scholar,16Gowda K. Zwieb C. Nucleic Acids Res. 1997; 25: 2835-2840Crossref PubMed Scopus (18) Google Scholar). RNA editing is a process that co or post-transcriptionally modifies RNA primary sequence from that encoded by the gene by either deletion, insertion, or base modification (17Smith H.C. Gott J.A. Hanson M.R. RNA. 1997; 3: 1105-1123PubMed Google Scholar). The phenomenon was first described in trypanosomatid mitochondrial mRNA and was shown to occur through a guide RNA-mediated cleavage and ligation (18Stuart K. Allen T.E. Kable M.L. Lawson S. Curr. Opin. Chem. Biol. 1997; 1: 340-346Crossref PubMed Scopus (15) Google Scholar). RNA editing has since been found in diverse lower and higher eukaryotes. The process can occur in the nucleus, chloroplast, and mito
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