Mapping of the Functional Boundaries and Secondary Structure of the Mouse Mammary Tumor Virus Rem-responsive Element
2009; Elsevier BV; Volume: 284; Issue: 38 Linguagem: Inglês
10.1074/jbc.m109.012476
ISSN1083-351X
AutoresJennifer A. Mertz, Amanda B. Chadee, Hyewon Byun, Rick Russell, Jaquelin P. Dudley,
Tópico(s)RNA and protein synthesis mechanisms
ResumoMouse mammary tumor virus (MMTV) is a complex retrovirus that encodes at least three regulatory and accessory proteins, including Rem. Rem is required for nuclear export of unspliced viral RNA and efficient expression of viral proteins. Our previous data indicated that sequences at the envelope-3′ long terminal repeat junction are required for proper export of viral RNA. To further map the Rem-responsive element (RmRE), reporter vectors containing various portions of the viral envelope gene and the 3′ long terminal repeat were tested in the presence and absence of Rem in transient transfection assays. A 476-bp fragment that spans the envelope-long terminal repeat junction had activity equivalent to the entire 3′-end of the mouse mammary tumor virus genome, but further deletions at the 5′- or 3′-ends reduced Rem responsiveness. RNase structure mapping of the full-length RmRE and a 3′-truncation suggested multiple domains with local base pairing and intervening single-stranded segments. A secondary structure model constrained by these data is reminiscent of the RNA response elements of other complex retroviruses, with numerous local stem-loops and long-range base pairs near the 5′- and 3′-boundaries, and differs substantially from an earlier model generated without experimental constraints. Covariation analysis provides limited support for basic features of our model. Reporter assays in human and mouse cell lines revealed similar boundaries, suggesting that the RmRE does not require cell type-specific proteins to form a functional structure. Mouse mammary tumor virus (MMTV) is a complex retrovirus that encodes at least three regulatory and accessory proteins, including Rem. Rem is required for nuclear export of unspliced viral RNA and efficient expression of viral proteins. Our previous data indicated that sequences at the envelope-3′ long terminal repeat junction are required for proper export of viral RNA. To further map the Rem-responsive element (RmRE), reporter vectors containing various portions of the viral envelope gene and the 3′ long terminal repeat were tested in the presence and absence of Rem in transient transfection assays. A 476-bp fragment that spans the envelope-long terminal repeat junction had activity equivalent to the entire 3′-end of the mouse mammary tumor virus genome, but further deletions at the 5′- or 3′-ends reduced Rem responsiveness. RNase structure mapping of the full-length RmRE and a 3′-truncation suggested multiple domains with local base pairing and intervening single-stranded segments. A secondary structure model constrained by these data is reminiscent of the RNA response elements of other complex retroviruses, with numerous local stem-loops and long-range base pairs near the 5′- and 3′-boundaries, and differs substantially from an earlier model generated without experimental constraints. Covariation analysis provides limited support for basic features of our model. Reporter assays in human and mouse cell lines revealed similar boundaries, suggesting that the RmRE does not require cell type-specific proteins to form a functional structure. Mouse mammary tumor virus (MMTV) 3The abbreviations used are: MMTVmouse mammary tumor virusDMRIE-C1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide-cholesterolGFPgreen fluorescent proteinHIV-1human immunodeficiency virus type 1LTRlong terminal repeatMOPS3-(N-morpholino)propanesulfonic acidRcRERec-responsive elementRemregulator of export/expression of MMTV mRNARmRERem-responsive elementRRERev-responsive elementSPsignal peptidentnucleotide(s)Sagsuperantigen. 3The abbreviations used are: MMTVmouse mammary tumor virusDMRIE-C1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide-cholesterolGFPgreen fluorescent proteinHIV-1human immunodeficiency virus type 1LTRlong terminal repeatMOPS3-(N-morpholino)propanesulfonic acidRcRERec-responsive elementRemregulator of export/expression of MMTV mRNARmRERem-responsive elementRRERev-responsive elementSPsignal peptidentnucleotide(s)Sagsuperantigen. has multiple regulatory and accessory genes (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar, 2Indik S. Günzburg W.H. Salmons B. Rouault F. Virology. 2005; 337: 1-6Crossref PubMed Scopus (82) Google Scholar). The known accessory genes specify a dUTPase (3Bergman A.C. Björnberg O. Nord J. Nyman P.O. Rosengren A.M. Virology. 1994; 204: 420-424Crossref PubMed Scopus (40) Google Scholar), which is believed to be involved in retroviral replication in non-dividing cells (4Payne S.L. Elder J.H. Curr. Protein Pept. Sci. 2001; 2: 381-388Crossref PubMed Scopus (50) Google Scholar), as well as superantigen (Sag). Sag is a transmembrane glycoprotein that is involved in the lymphocyte-mediated transmission of MMTV from maternal milk in the gut to susceptible epithelial cells in the mammary gland (5Golovkina T.V. Chervonsky A. Dudley J.P. Ross S.R. Cell. 1992; 69: 637-645Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 6Held W. Waanders G.A. Shakhov A.N. Scarpellino L. Acha-Orbea H. MacDonald H.R. Cell. 1993; 74: 529-540Abstract Full Text PDF PubMed Scopus (185) Google Scholar). The Sag protein expressed by endogenous (germline) MMTV proviruses has been reported to provide susceptibility to infection by exogenous MMTVs or the bacterial pathogen, Vibrio cholerae (7Bhadra S. Lozano M.M. Payne S.M. Dudley J.P. PLoS Pathog. 2006; 2: e128Crossref PubMed Scopus (21) Google Scholar). These results suggest a role for MMTV Sag in the host innate immune response. mouse mammary tumor virus 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide-cholesterol green fluorescent protein human immunodeficiency virus type 1 long terminal repeat 3-(N-morpholino)propanesulfonic acid Rec-responsive element regulator of export/expression of MMTV mRNA Rem-responsive element Rev-responsive element signal peptide nucleotide(s) superantigen. mouse mammary tumor virus 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide-cholesterol green fluorescent protein human immunodeficiency virus type 1 long terminal repeat 3-(N-morpholino)propanesulfonic acid Rec-responsive element regulator of export/expression of MMTV mRNA Rem-responsive element Rev-responsive element signal peptide nucleotide(s) superantigen. MMTV recently was shown to be a complex retrovirus (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). Complex retroviruses encode RNA-binding proteins that facilitate nuclear export of unspliced viral RNA by using a leucine-rich nuclear export sequence (8Malim M.H. Emerman M. Cell Host Microbe. 2008; 3: 388-398Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar), which binds to chromosome region maintenance 1 (Crm1)(9Neville M. Stutz F. Lee L. Davis L.I. Rosbash M. Curr. Biol. 1997; 7: 767-775Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), whereas simple retroviruses have a cis-acting constitutive transport element that directly interacts with components of the Tap/NXF1 pathway (10Grüter P. Tabernero C. von Kobbe C. Schmitt C. Saavedra C. Bachi A. Wilm M. Felber B.K. Izaurralde E. Mol. Cell. 1998; 1: 649-659Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). Similar to other complex retroviruses, MMTV encodes a Rev-like protein, regulator of export/expression of MMTV mRNA (Rem) (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). Rem is translated from a doubly spliced mRNA into a 33-kDa protein that contains nuclear and nucleolar localization signals as well as a predicted RNA-binding motif and leucine-rich nuclear export sequence (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar, 2Indik S. Günzburg W.H. Salmons B. Rouault F. Virology. 2005; 337: 1-6Crossref PubMed Scopus (82) Google Scholar). Our previous experiments indicated that Rem affects export of unspliced viral RNA, and a reporter vector that relies on luciferase expression from unspliced RNAs has increased activity in the presence of Rem (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). Sequences at the MMTV envelope-long terminal repeat (LTR) junction were required within the vector for Rem-induced expression, suggesting that the LTR contains all or part of the Rem-responsive element (RmRE). Very recently, Müllner et al. (11Müllner M. Salmons B. Günzburg W.H. Indik S. Nucleic Acids Res. 2008; 36: 6284-6294Crossref PubMed Scopus (29) Google Scholar) identified a 490-nt region spanning the MMTV envelope-3′ LTR region, which was predicted to form a highly structured RNA element. This element confers Rem responsiveness on heterologous human immunodeficiency virus type 1 (HIV-1)-based plasmid constructs in transfection experiments. Experiments using other retroviral export proteins have demonstrated considerable variation in the size of the response elements. A minimal Rev-responsive element (RRE) in the human immunodeficiency virus type 1 (HIV-1) genomic RNA is 234 nt, the human T-cell leukemia virus Rex-responsive element is 205 nt (12Ahmed Y.F. Hanly S.M. Malim M.H. Cullen B.R. Greene W.C. Genes Dev. 1990; 4: 1014-1022Crossref PubMed Scopus (78) Google Scholar, 13Toyoshima H. Itoh M. Inoue J. Seiki M. Takaku F. Yoshida M. J. Virol. 1990; 64: 2825-2832Crossref PubMed Google Scholar, 14Hanly S.M. Rimsky L.T. Malim M.H. Kim J.H. Hauber J. Duc Dodon M. Le S.Y. Maizel J.V. Cullen B.R. Greene W.C. Genes Dev. 1989; 3: 1534-1544Crossref PubMed Scopus (134) Google Scholar), whereas the Rec-responsive element (RcRE; also known as the K-RRE) of human endogenous retrovirus type K is 416 to 429 nt (15Yang J. Bogerd H. Le S.Y. Cullen B.R. RNA. 2000; 6: 1551-1564Crossref PubMed Scopus (26) Google Scholar, 16Magin-Lachmann C. Hahn S. Strobel H. Held U. Löwer J. Löwer R. J. Virol. 2001; 75: 10359-10371Crossref PubMed Scopus (38) Google Scholar). Most response elements are confined to the 3′-end of their respective retroviral genomes (either to the envelope or LTR regions) (14Hanly S.M. Rimsky L.T. Malim M.H. Kim J.H. Hauber J. Duc Dodon M. Le S.Y. Maizel J.V. Cullen B.R. Greene W.C. Genes Dev. 1989; 3: 1534-1544Crossref PubMed Scopus (134) Google Scholar, 15Yang J. Bogerd H. Le S.Y. Cullen B.R. RNA. 2000; 6: 1551-1564Crossref PubMed Scopus (26) Google Scholar), but 5′ Rev-response elements also have been identified (17Groom H.C. Anderson E.C. Dangerfield J.A. Lever A.M. J. Gen. Virol. 2009; 90: 1141-1147Crossref PubMed Scopus (34) Google Scholar). Studies indicate that the secondary structure is a critical factor for proper function of retroviral response elements (18Holland S.M. Ahmad N. Maitra R.K. Wingfield P. Venkatesan S. J. Virol. 1990; 64: 5966-5975Crossref PubMed Google Scholar), and that multiple stem-loops are required. Export proteins multimerize on these elements to allow activity (19Askjaer P. Jensen T.H. Nilsson J. Englmeier L. Kjems J. J. Biol. Chem. 1998; 273: 33414-33422Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). In the current study, we have used deletion mutations within a reporter vector based on the 3′-end of the MMTV genome to define a 476-nt element necessary for maximum Rem responsiveness. This element spans the envelope-LTR junction of the MMTV genome as previously reported (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). However, a secondary structure model generated using digestions of the RmRE by RNases V1, T1, and A as experimental constraints differs significantly from the published structure (11Müllner M. Salmons B. Günzburg W.H. Indik S. Nucleic Acids Res. 2008; 36: 6284-6294Crossref PubMed Scopus (29) Google Scholar) and more closely resembles complex retroviral response elements. Transfection experiments indicated that the MMTV RmRE could function in both mouse and human cells, suggesting that conserved cellular proteins interact with Rem. XC rat fibroblast cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, gentamicin sulfate (50 μg/ml), penicillin (100 units/ml), and streptomycin (50 μg/ml). Jurkat human T lymphoma cells were maintained in RPMI media supplemented with 5% fetal calf serum, gentamicin sulfate (50 μg/ml), penicillin (100 units/ml), and streptomycin (50 μg/ml). XC cells were transfected in 6-well plates using DMRIE-C transfection reagent (Invitrogen) according to the manufacturer's instructions. Each transfection contained 250 ng of the reporter vector, 250 ng of pGL3-control plasmid, and 2 μg of an expression vector for Rem or green fluorescent protein (GFP). Jurkat cells were transfected by electroporation using a BTX ECM600 instrument. Cells (1 × 107) were mixed with the appropriate plasmid DNA in a volume of 400 μl of Jurkat medium prior to electroporation in 4-mm gap cuvettes (260 V, 1050 microfarads, and 720 ohms). Each transfection contained 1 μg of the reporter vector, 1 μg of the GL3-control plasmid, and 20 μg of expression vectors for Rem or GFP. Transfected cells were incubated at 37 °C in complete medium and harvested 2 days after transfection. All transfections were performed in triplicate using the same amount of total DNA in each sample. Cytoplasmic extracts obtained by three freeze-thaw cycles were stored at −70 °C prior to luciferase assays. The plasmids GFP-Rem (RemP71), HMRluc, and HMΔeLTRluc have been described elsewhere (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). The plasmid pEGFPN3 was obtained from Clontech and the pGL3-control plasmid was obtained from Promega. The mutant HMΔeLTR+XRluc constructs were made by insertion of the mutant RmRE (X) into an engineered ScaI site downstream of the splice acceptor site and upstream of the simian virus 40 poly(A) signal in HMΔeLTRluc (Fig. 1). In vitro transcription vectors for wild-type and mutant RmREs were made by PCR amplification of the wild-type or mutant RmRE with insertion of a T7 polymerase promoter upstream of the RmRE. The PCR product was then inserted into the multiple cloning site of the pEGFPN3 vector. The 1–348 mutant containing the 3′-truncated RmRE was amplified with the following primers: T7HM+, 5′-TAA TAC GAC TCA CTA TAG GGA TCT TAA CGT GCT TC-3′ and RmRE 348, 5′-AGT ACT GTG GTC CTT GCC TCA GGA GG-3′. Cloning of the GFP-RemP71L expression vector and all other mutant RmRE constructs was performed using site-directed mutagenesis or by cleavage with restriction enzymes and religation. Details are available upon request. All constructs were confirmed by automated sequencing reactions. Luciferase assays were performed using the Dual Luciferase Reporter Assay system (Promega) to quantitate both Renilla and firefly luciferase activities (20Mertz J.A. Mustafa F. Meyers S. Dudley J.P. J. Virol. 2001; 75: 2174-2184Crossref PubMed Scopus (20) Google Scholar). A firefly luciferase reporter vector lacking MMTV sequences was added to each transfection, and activities showed that different transfections within the same experiment had similar DNA uptake. Samples of normalized and unnormalized data have been provided (see supplemental Table S1 compared with Fig. 2). Protein extracts for Western blotting were obtained as previously described (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). Western blots were performed using antibodies specific for the GFP tag (Clontech) on GFP-Rem or actin (Calbiochem); the latter served as a control for protein loading. Proteins were detected using the ECL Western blotting detection system (Amersham Biosciences). DNAs encoding the wild-type and truncated RmREs (1–496 and 1–348) were linearized using ScaI (New England Biolabs). RNAs were prepared by in vitro transcription using T7 RNA polymerase and purified using a Qiagen RNeasy column following the manufacturer's protocol. RNAs were incubated with shrimp alkaline phosphatase (Promega) to remove the 5′-triphosphate and then with [γ-32P]ATP (PerkinElmer Life Sciences) and T4 polynucleotide kinase (New England Biolabs) to add a radiolabeled phosphoryl group to the 5′-end of the RNA (21Donis-Keller H. Maxam A.M. Gilbert W. Nucleic Acids Res. 1977; 4: 2527-2538Crossref PubMed Scopus (1039) Google Scholar). RNAs were 3′-end labeled using [5′-32P]pCp and T4 RNA ligase to extend the 3′-end of the RNA with a single-labeled nucleotide (22England T.E. Uhlenbeck O.C. Nature. 1978; 275: 560-561Crossref PubMed Scopus (415) Google Scholar). Both 3′- and 5′-labeled RNAs were purified using 8% native PAGE. RNase footprinting reactions (10 μl) were performed using in vitro transcribed full-length (1–496) or 3′-truncated (1–348) RmRE RNA. Labeled RNA (3–15 nm, 10,000 cpm/μl) was incubated in 50 mm sodium/MOPS (pH 7.0) and 10 mm MgCl2. Varying the preincubation time did not affect the results (15 s to 5 min), suggesting that the RNA formed the secondary structure rapidly and stably. Nucleases were then added at levels empirically determined to give optimal RNA cleavage: 0.1 unit of RNase T1 (Sigma), 10−4 units of RNase V1 (Ambion), and 10−3 units of RNase A (Sigma). RNA was digested for 1 to 3 min at 25 °C, and aliquots were quenched by adding 1 mg/ml proteinase K and then 2 volumes of 20 mm EDTA in loading dye (90% (v/v) formamide, 0.04% xylene cyanol, and 0.04% bromphenol blue). Reaction tubes were immediately placed in liquid nitrogen to ensure that the RNases were fully inactivated. RNA sequencing ladders were generated by digesting 5′- or 3′-labeled RNA with 0.1 unit of RNase T1 for 15 min under denaturing conditions (7 m urea at 50 °C). Reaction products were separated by 8% denaturing PAGE and quantitated using Semi-Automated Footprinting Analysis software (23Das R. Laederach A. Pearlman S.M. Herschlag D. Altman R.B. RNA. 2005; 11: 344-354Crossref PubMed Scopus (255) Google Scholar). From each digestion, raw intensity values corresponding to cleavage at each nucleotide were normalized by dividing each value by the average intensity of all bands within the range quantitated. These normalized values were then averaged between experimental determinations to produce final values. Except where indicated, all reported results reflect the averages of two to six independent determinations. To determine the boundaries of the MMTV RmRE, several constructs were designed based on our previously described pHMRluc vector (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar, 24Mertz J.A. Lozano M.M. Dudley J.P. Retrovirology. 2009; 6: 10Crossref PubMed Scopus (28) Google Scholar). This vector contains the 3′-end of the MMTV genome, including part of the envelope gene and the 3′ LTR, downstream of the cytomegalovirus promoter (Fig. 1). Because the Renilla luciferase gene was inserted between the splice donor and acceptor sites in the envelope gene, detection of luciferase activity in transfected cells indicates export of unspliced mRNA from the nucleus to the cytoplasm. Previous data showed that reporter gene activity from this vector is induced by co-expression of the MMTV export protein, Rem, in trans (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). Rem-induced luciferase activity also required the presence of the envelope-3′ LTR junction in the reporter vector, suggesting that these sequences contain the RmRE (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). The pHMΔeLTRluc plasmid, which substitutes the simian virus 40 polyadenylation region for the MMTV 3′ LTR (Fig. 1), shows no response to the addition of Rem expression vectors (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). Therefore, various MMTV sequences at the envelope-LTR border were re-inserted into the pHMΔeLTRluc vector to determine the region necessary for Rem responsiveness. Our previous experiments showed that insertion of a BglII to ScaI fragment (496 bp) within the pHMΔeLTRluc plasmid gave the same Rem response in mouse cells as the wild-type pHMRluc vector, which contains the entire 3′-end of the MMTV genome (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar). In agreement with these results, both reporter plasmids showed about 5-fold increases in luciferase activity after co-expression of Rem in transient transfections of XC rat cells (compare pHMRluc to the 1–496 vector in Fig. 2A). Unlike mouse cells, XC rat cells lack endogenous Mtv proviruses, which may express virally encoded proteins (7Bhadra S. Lozano M.M. Payne S.M. Dudley J.P. PLoS Pathog. 2006; 2: e128Crossref PubMed Scopus (21) Google Scholar). Western blotting was also used to verify expression of the Rem construct (data not shown). Subsequent plasmids were designed to determine the 5′ border of the response element (RmRE). Deletion of 10 or 20 nt from the RmRE 5′-end had no reproducible effect on Rem responsiveness (constructs 11–496 and 21–496) (see Fig. 2A and Table 1). However, reporter plasmids with a deletion of 30 bp from the 5′-end of the vector showed a 2.5-fold decrease in reporter levels after Rem expression, and constructs with a deletion of 40 bp or more had little or no detectable Rem response (see Fig. 2A and Table 1).TABLE 1Reporter gene activity of RmRE deletion and substitution mutantsRmRE constructRelative Rem responseaAverage luciferase activities of triplicate mutant transfections in XC cells are given relative to the wild-type (WT) RmRE construct (±S.D.).%Wild-type (1–496)100 ± 14bThe Rem-dependent increase for the wild-type plasmid was 5–7-fold, which was designated as 100%.11–496110 ± 2121–496108 ± 3831–49639 ± 7.141–496055–49601–39850 ± 4.01–37351 ± 111–34821 ± 4.7Δ50–3698.9 ± 0.2a Average luciferase activities of triplicate mutant transfections in XC cells are given relative to the wild-type (WT) RmRE construct (±S.D.).b The Rem-dependent increase for the wild-type plasmid was 5–7-fold, which was designated as 100%. Open table in a new tab Deletions also were performed to remove sequences from the 3′-end of the RmRE. Removal of 98 or 123 bp at the 3′-end of the RmRE of the reporter vector resulted in a 2-fold drop in Rem responsiveness, and deletion of 148 bp essentially showed no Rem response (Fig. 2B). Furthermore, a reporter construct carrying an internal deletion of the RmRE between 50 and 369 bp (Δ50–369) had little detectable Rem response (Table 1). These results suggest that full Rem responsiveness requires the majority of the BglII-ScaI fragment spanning the envelope-3′ LTR region. After defining the functional boundaries of the RmRE, we used RNase mapping to probe its secondary structure. RNA spanning the wild-type C3H MMTV RmRE (nt 1–496) was produced by in vitro transcription, 5′- or 3′-end labeled with 32P, and subjected to limited digestion with RNases T1, A, and V1. RNase T1 cleaves preferentially at single-stranded G residues, RNase A cleaves at single-stranded C or U residues, and RNase V1 primarily digests base-paired nucleotides without strong preferences for nucleotide identity (25Ehresmann C. Baudin F. Mougel M. Romby P. Ebel J.P. Ehresmann B. Nucleic Acids Res. 1987; 15: 9109-9128Crossref PubMed Scopus (660) Google Scholar). Thus, RNase probes used in combination give extensive information on base pairing status throughout a structured RNA (26Kjems J. Brown M. Chang D.D. Sharp P.A. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 683-687Crossref PubMed Scopus (185) Google Scholar, 27Fuchs G. Stein A.J. Fu C. Reinisch K.M. Wolin S.L. Nat. Struct. Mol. Biol. 2006; 13: 1002-1009Crossref PubMed Scopus (62) Google Scholar). Results from digestions with each RNase are shown for a portion of the RmRE (Fig. 3). The RNA includes extensive secondary structure, as indicated by localized regions that were cleaved efficiently by RNase V1 and were inaccessible to RNases A and T1 (e.g. nt 199–200 and 231–234) (Fig. 3A). On the other hand, these segments were limited to a few consecutive nucleotides and were interrupted by local regions of accessibility to the single strand-specific RNases, suggesting a structure in which short helical segments are separated by many hairpin and/or internal loops. To evaluate the RNase mapping results quantitatively, we used the freely available software Semi-Automated Footprinting Analysis (23Das R. Laederach A. Pearlman S.M. Herschlag D. Altman R.B. RNA. 2005; 11: 344-354Crossref PubMed Scopus (255) Google Scholar) to determine the intensity of RNase-mediated cleavage at each position. Digestion profiles for the three RNases are provided (Fig. 3B) across the range shown in the gel (Fig. 3A). Profiles for the entire RmRE also are shown (supplemental Figs. S1–S3). From the average profiles of multiple independent determinations, we established empirically two threshold levels of intensity. Bands that significantly exceeded the average band intensity were considered to reflect nucleotides that were accessible to each nuclease. A second, higher threshold was also established to separate the smaller groups of nucleotides that were the most accessible to each RNase probe (supplemental Figs. S1–S3). With the latter set of the most reproducible and largest signals as experimental constraints, we used Mfold to generate possible secondary structures of the RmRE (28Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10157) Google Scholar). The structure predicted to be most stable, after using constraints from the RNase digestions, is shown (Fig. 4). This structure is highly complex, containing many single-stranded regions and multiple hairpin loops typical of the response elements of other complex retroviruses (12Ahmed Y.F. Hanly S.M. Malim M.H. Cullen B.R. Greene W.C. Genes Dev. 1990; 4: 1014-1022Crossref PubMed Scopus (78) Google Scholar, 26Kjems J. Brown M. Chang D.D. Sharp P.A. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 683-687Crossref PubMed Scopus (185) Google Scholar, 29Malim M.H. Hauber J. Le S.Y. Maizel J.V. Cullen B.R. Nature. 1989; 338: 254-257Crossref PubMed Scopus (945) Google Scholar, 30Legiewicz M. Badorrek C.S. Turner K.B. Fabris D. Hamm T.E. Rekosh D. Hammarskjöld M.L. Le Grice S.F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 14365-14370Crossref PubMed Scopus (46) Google Scholar, 31Askjaer P. Kjems J. J. Biol. Chem. 1998; 273: 11463-11471Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). This structure has been divided into regions I, II, III, and IV. Also similar to other response elements, the secondary structure models favored by Mfold include long-range base pairings between sequences close to the 5′ and 3′ boundaries of the RmRE within region I (Fig. 4 and data not shown). Interestingly, in the absence of experimental constraints, Mfold predicted secondary structures that differed substantially from that shown in Fig. 4. The most favorable structure in the absence of the constraints is shown for comparison (see supplemental Fig. S4). Although this structure retains the most basic features of the model in Fig. 4, multiple stem-loops and long-range pairings between sequences near the boundaries, local interactions differ substantially. Importantly, the model generated by including experimental constraints provides better agreement with the footprinting data that were not used as constraints. Of 43 nucleotides that were reactive to RNase V1, but were not constrained in the structural prediction, 72% are correctly predicted to be double-stranded, compared with only 35% for the unconstrained model (data not shown). The agreement with RNase A data were comparable for the two models (41% of reactive nucleotides are single-stranded in the model generated with constraints versus 35% for the model without constraints), whereas, for RNase T1, only six reactive nucleotides were not used as constraints, preventing meaningful analysis. When all of the footprinting data are considered, the constrained model gives much higher rates of agreement for all three RNases. Although this is true by necessity because many of the reactive nucleotides were constrained in the modeling, this comparison nevertheless highlights the point that a secondary structure may be obtained to give much stronger agreement with experimental results than the one that would be favored in the absence of experimental constraints. Previous experiments suggest that Rem is synthesized as a 33-kDa precursor protein, which is targeted by an unusually long signal peptide (SP) (98 amino acids) for translocation across the endoplasmic reticulum membrane (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Scholar, 32Dultz E. Hildenbeutel M. Martoglio B. Hochman J. Dobberstein B. Kapp K. J. Biol. Chem. 2008; 283: 9966-9976Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Subsequently, the Rem C terminus is glycosylated, and the N-terminal SP appears to be cleaved by signal peptidase in the endoplasmic reticulum lumen. Our previous data showed that GFP-tagged Rem accumulates in nucleoli (1Mertz J.A. Simper M.S. Lozano M.M. Payne S.M. Dudley J.P. J. Virol. 2005; 79: 14737-14747Crossref PubMed Scopus (99) Google Sch
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