Structural Dynamics of HIV-1 Rev and Its Complexes with RRE and 5S RNA

Lam, Wai-Chung; Seifert, Jan; Amberger, F.; Gräf, Christine; Auer, Manfred; Millar, David P.

doi:10.1021/bi9719096

Cited by 21 publications

(21 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As shown in a previous study by time-re-2 O. Rosorius, unpublished data. solved fluorescence spectroscopy, Rev appears to interact with 5 S rRNA in a similar manner to its interaction with its homologous viral Rev response element RNA target (30). In close agreement with these data, GST-Rev protein clearly bound 5 S rRNA in our gel retardation assay system (Fig.…”

Section: Nuclear Import and Nucleolar Localization Of Ribosomalsupporting

confidence: 87%

See 1 more Smart Citation

Human Ribosomal Protein L5 Contains Defined Nuclear Localization and Export Signals

Rosorius

Fries

Stauber

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

Ribosomal protein L5 is part of the 60 S ribosomal subunit and localizes in both the cytoplasm and the nucleus of eukaryotic cells, accumulating particularly in the nucleoli. L5 is known to bind specifically to 5 S rRNA and is involved in nucleocytoplasmic transport of this rRNA. Here, we report a detailed analysis of the domain organization of the human ribosomal protein L5. We show that a signal that mediates nuclear import and nucleolar localization maps to amino acids 21-37 within the 297-amino acid L5 protein. Furthermore, carboxyl-terminal residues at positions 255-297 serve as an additional nuclear/nucleolar targeting signal. Domains involved in 5 S rRNA binding are located at both the amino terminus and the carboxyl terminus of L5. Microinjection studies in somatic cells demonstrate that a nuclear export signal (NES) that maps to amino acids 101-111 resides in the central region of L5. This NES is characterized by a pronounced clustering of critical leucine residues, which creates a peptide motif not previously observed in other leucine-rich NESs. Finally, we present a refined model of the multidomain structure of human ribosomal protein L5.The biogenesis of eukaryotic ribosomes occurs at a specific subnuclear compartment, the nucleolus, and requires the coordinated assembly of four different rRNAs and approximately 80 ribosomal proteins (1, 2). The 5.8, 18, and 28 S rRNAs are synthesized by RNA polymerase I in the nucleolus, whereas, in contrast, 5 S rRNA is transcribed by RNA polymerase III in the nucleoplasm. The ribosomal proteins are encoded by mRNAs that are synthesized by RNA polymerase II. After translation, these proteins are imported from the cytoplasm into the nucleolus for assembly of the 40 S and 60 S ribosomal subunits. These, in turn, are then exported to the cytoplasm. Thus, multiple intracellular transport activities between the nucleus and cytoplasm are required for de novo ribosome synthesis.In eukaryotic cells, the 5 S rRNA is part of the 60 S ribosomal subunit. In addition, a significant amount of 5 S rRNA is complexed with various proteins to form nonribosome-associated ribonucleoprotein particles. After transcription, 5 S rRNA is able to transiently bind the La antigen, a 50-kDa protein that acts in the termination of polymerase III transcripts, in the nucleus (3, 4). Furthermore, in the nucleus, 5 S rRNA also binds either its own transcription factor IIIA or ribosomal protein L5, forming 7 or 5 S ribonucleoprotein particles, respectively (reviewed in Ref. 5). In particular, it has been suggested that the 5 S rRNA⅐L5 complex (5 S ribonucleoprotein particle) acts as a precursor to ribosome assembly by delivering 5 S rRNA from the nucleoplasm to the nucleolar assembly site of 60 S ribosomal subunits (6). Studies in Xenopus oocytes have shown that 5 S rRNA can be exported from the nucleus to the cytoplasm for subsequent accumulation at distinct cytoplasmic storage sites by either transcription factor IIIA or L5 (7,8). As a consequence of increased ribosomal subunit synthesis, stored 5...

show abstract

Section: Nuclear Import and Nucleolar Localization Of Ribosomalsupporting

confidence: 87%

“…Summarizing the data obtained in living and fixed cells, strong nuclear import of L5 is mediated by a region located in the amino terminus of the protein (aa [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. This sequence also contributes to nucleolar localization of L5.…”

Section: Nuclear Import and Nucleolar Localization Of Ribosomalmentioning

confidence: 99%

Human Ribosomal Protein L5 Contains Defined Nuclear Localization and Export Signals

Rosorius

Fries

Stauber

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Since import of HIV-1 Rev appears to be directed by a NLS resembling the SV40 T-antigen NLS and also uses the importin b-pathway [Henderson and Percipalle, 1997] the effects of transcription inhibitors on traf®cking could also be indirect. Preventing active transcription might affect secondary binding sites in the nucleus (e.g., by the degradation of 5S RNA at the nucleolus which binds the HIV-1 Rev protein [Lam et al, 1998] and therefore changes the steady-state localization of the protein, rather than interfering directly with transport. Additional studies in de®ned import/export systems are necessary to completely understand the effect of transcription inhibitors on the traf®cking of HIV-1 Rev, VHL or PABP1.…”

Section: Resultsmentioning

confidence: 99%

Investigation of nucleo-cytoplasmic transport using UV-guided microinjection

et al. 2000

View full text Add to dashboard Cite

Active nucleo-cytoplasmic transport is mediated by dynamic signal-mediated pathways. We investigated the effects of transcription inhibitors or fluorescent lectins on nuclear import mediated by nuclear localization signals (NLSs). Therefore, a novel experimental approach that allows the controlled sequential introduction of fluorescent substances into living cells was established. A microinjection system equipped with an UV-source enabled us to identify fluorescent-labeled cells for the subsequent introduction of additional fluorescent compounds, in order to study their interactions in vivo. Cells were initially labeled either by expression of autofluorescent proteins or by microinjection of fluorescent substances. Transcription inhibitors did not affect nuclear transport mediated by classical NLSs but inhibited import mediated by the M9-domain of hnRNPA1. Comparison of a mono- and bipartite NLS revealed that the bipartite signal was more active in import. Sequential injection of differentially labeled nuclear import and export substrates allowed monitoring of import and export simultaneously in the same living cell. The introduced experimental approach will also be useful to analyze a variety of biological processes in living mammalian cells.

show abstract

“…Several lines of evidence suggest that Rev assembles into the Rev-RRE complex in a sequential manner (Lam et al, 1998; Van Ryk and Venkatesan, 1999; Daugherty et al, 2008, 2010a; Pond et al, 2009; Robertson-Anderson et al, 2011). Some evidence suggests that Rev is recruited to the RRE one molecule at a time (Pond et al, 2009), while other data are consistent with recruitment in the form of a dimeric complex (Daugherty et al, 2008, 2010b).…”

Section: Introductionmentioning

confidence: 99%

RNA-guided assembly of Rev-RRE nuclear export complexes

Bai

Tambe

Zhou

et al. 2014

eLife

140

View full text Add to dashboard Cite

HIV replication requires nuclear export of unspliced and singly spliced viral transcripts. Although a unique RNA structure has been proposed for the Rev-response element (RRE) responsible for viral mRNA export, how it recruits multiple HIV Rev proteins to form an export complex has been unclear. We show here that initial binding of Rev to the RRE triggers RNA tertiary structural changes, enabling further Rev binding and the rapid formation of a viral export complex. Analysis of the Rev-RRE assembly pathway using SHAPE-Seq and small-angle X-ray scattering (SAXS) reveals two major steps of Rev-RRE complex formation, beginning with rapid Rev binding to a pre-organized region presenting multiple Rev binding sites. This step induces long-range remodeling of the RNA to expose a cryptic Rev binding site, enabling rapid assembly of additional Rev proteins into the RNA export complex. This kinetic pathway may help maintain the balance between viral replication and maturation.DOI: http://dx.doi.org/10.7554/eLife.03656.001

show abstract

Structural Dynamics of HIV-1 Rev and Its Complexes with RRE and 5S RNA

Cited by 21 publications

References 54 publications

Human Ribosomal Protein L5 Contains Defined Nuclear Localization and Export Signals

Human Ribosomal Protein L5 Contains Defined Nuclear Localization and Export Signals

Investigation of nucleo-cytoplasmic transport using UV-guided microinjection

RNA-guided assembly of Rev-RRE nuclear export complexes

Contact Info

Product

Resources

About