Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Apr;16(4):817-27.
doi: 10.1261/rna.1712910. Epub 2010 Feb 24.

LSm1-7 complexes bind to specific sites in viral RNA genomes and regulate their translation and replication

Rui Pedro Galão  1 , Ashwin Chari, Isabel Alves-Rodrigues, Daniela Lobão, Antonio Mas, Christian Kambach, Utz Fischer, Juana Díez
Affiliations

LSm1-7 complexes bind to specific sites in viral RNA genomes and regulate their translation and replication

Rui Pedro Galão et al. RNA. 2010 Apr.

Abstract

LSm1-7 complexes promote cellular mRNA degradation, in addition to translation and replication of positive-strand RNA viruses such as the Brome mosaic virus (BMV). Yet, how LSm1-7 complexes act on their targets remains elusive. Here, we report that reconstituted recombinant LSm1-7 complexes directly bind to two distinct RNA-target sequences in the BMV genome, a tRNA-like structure at the 3'-untranslated region and two internal A-rich single-stranded regions. Importantly, in vivo analysis shows that these sequences regulate the translation and replication of the BMV genome. Furthermore, both RNA-target sequences resemble those found for Hfq, the LSm counterpart in bacteria, suggesting conservation through evolution. Our results provide the first evidence that LSm1-7 complexes interact directly with viral RNA genomes and open new perspectives in the understanding of LSm1-7 functions.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
The BMV genome and the initial BMV replication steps. (A) Schematic diagram of the BMV tripartite genome showing the ORFs (solid black boxes) and the untranslated regions (UTRs) (single lines). The three BMV RNAs are capped (m7G) and end in a tRNA-like structure (cloverleaf structure). The location of the recruitment element (RE) is shown for the three RNAs. (B) After translation of the viral proteins, the 1a helicase recognizes the RE sequence to specifically recruit the viral genomes from the cellular translation machinery to viral-induced invaginations in the endoplasmic reticulum where replication occurs. The LSm1-7 complex plays a key role in the regulation of these processes.
FIGURE 2.
FIGURE 2.
A 3′poly(A) tail suppresses the requirement of LSm1-7 complexes for RNA3 translation. (A) Schematic representation of the RNA3 and the RNA3-poly(A) derivative [RNA3p(A)] in which the 3′ UTR was replaced by the yeast ADH1 polyadenylation site. (B) WT and lsm1Δ yeast strains were transformed with a plasmid harboring RNA3 or RNA3p(A). (Upper panels) Western blot analysis of the 3a protein expression. As a control for equal loading of total protein, expression of the phosphoglycerate kinase protein (PGK) was also analyzed. (Lower panels) Northern blot analysis of RNA3 accumulation. Detection of the 18S ribosomal RNA was used to assure equal loading and sample quality. Histograms show the average and standard error of the mean of the accumulation of 3a protein relative to the amount of RNA3 for at least three independent colonies. The average value obtained for RNA3 in WT cells was set to 100.
FIGURE 3.
FIGURE 3.
Binding of LSm1-7 complexes to the 3′ UTRs of the three BMV RNAs depends on a tRNA-like structure. (A) Coomassie blue-stained SDS-polyacrylamide gel of purified reconstituted LSm1-7 and LSm2-8 complexes (left). Binding specificity of LSm1-7 and LSm2-8 complexes were determined by gel-shift analysis using recombinant rings and radiolabeled gel-purified U1 and U6 snRNAs (right). After complex formation, samples were loaded on a nondenaturating polyacrylamide gel and visualized by autoradiography. (B) Schematic representation of generated transcripts. The numeration refers to the position of the corresponding nucleotides in the complete BMV RNA genomes. (C,D) Gel-shift assays were performed as described above. LSm1-7 complexes were incubated with radiolabeled transcribed RNAs corresponding to the 5′ and 3′ UTRs (C) or with the 3′ UTR TLS and NTLS sequences of the three genomic BMV RNAs (D). The plant tRNA corresponds to the Tyr-tRNA of Nicotania bentamiana. Asterisks indicate the position of the gel-shifted RNAs.
FIGURE 4.
FIGURE 4.
The complete 3′ UTR is required for optimal interaction with the LSm1-7 complexes. (A) Secondary structure of the TLS from the 3′ UTR of BMV RNA3. Numbers refer to corresponding nucleotide positions in the BMV RNA3 genome. The various stem–loop structures are indicated (A–E). To facilitate their identification some of them are highlighted with different shadowing (data adapted from Barends et al. 2004). (B) RNA substrates corresponding to the NTLS region of the RNA3 3′ UTR and to the same sequence plus consecutive TLS domains or (C) RNA substrates corresponding to the TLS region of the RNA3 3′ UTR, and to the same sequence plus additional stretches from the NTLS region were incubated with purified LSm1-7 complexes and analyzed by gel-shift assays as described in Figure 3.
FIGURE 5.
FIGURE 5.
Binding of LSm1-7 complexes to the intergenic region (IR) of RNA3 depends on the A-rich loops L1 and L2. (A) Secondary structure of the IR of BMV RNA3. Numbers refer to the corresponding nucleotide positions in the BMV RNA3 genome. The recruitment element and the loops L1 and L2 are shown. In bold are indicated the stop codon of the 3a ORF and the start codon of the coat protein that limit the IR. (B) Schematic diagram of the generated RNA transcripts used to map the sequences in the IR that mediate the interaction with the LSm1-7 complexes. (C) The corresponding radiolabeled RNAs were used to perform gel-shift assays with LSm1-7 complexes as described in Figure 3.
FIGURE 6.
FIGURE 6.
LSm1-7 complexes bind to the 3′ UTR and the IR specifically and through a common binding site. Radiolabeled RNA3 3′ UTR (upper panels) and RNA3 IR (lower panels) were incubated with reconstituted LSm1-7 complexes in the presence of increasing amounts of unlabeled RNA1 5′ UTR, RNA3 3′ UTR, or RNA3 IR transcripts as competitors.
FIGURE 7.
FIGURE 7.
Deletion of loops L1 and L2 in the IR inhibits translation and favors recruitment of BMV RNA3. (A) Generated BMV RNA3 derivatives in which the internal loop1 (ΔL1), loop2 (ΔL2), or both (ΔL1L2), were deleted. (B) WT and lsm1Δ strains were transformed with a plasmid harboring the RNA3 or the RNA3 derivatives (ΔL1, ΔL2, or ΔL1L2) and the accumulations of the 3a protein and RNA3 were analyzed by Western and Northern blotting, respectively, as in Figure 2. Histograms show the average and standard error of the mean of the accumulation of 3a protein relative to the amount of RNA3 from at least three independent colonies. The average 3a/RNA3 value in WT yeast was set to 100. (C) WT and lsm1Δ yeast strains were transformed with a plasmid expressing the 1a protein plus a plasmid transcribing the WT RNA3 or the RNA3 derivatives (ΔL1, ΔL2, or ΔL1L2). RNA3 accumulation was analyzed by Northern blotting and 1a protein expression by Western blotting. Histograms represent relative accumulation of RNA3 and the RNA3 derivatives in WT and lsm1Δ cells of three independent colonies. The average accumulation of RNA3 in WT yeast was set to 100.
FIGURE 8.
FIGURE 8.
Model of LSm1-7 function on BMV RNA3. Binding of LSm1-7 complexes to the tRNA-like structure in the 3′ UTR and the internal loops in the IR would regulate translation and recruitment to replication of BMV RNA3. Given that the LSm1-7 ring does not bind simultaneously to both RNA sequences, the interaction with two rings might be considered.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ahlquist P. Bromovirus RNA replication and transcription. Curr Opin Genet Dev. 1992;2:71–76. - PubMed
    1. Ahola T, den Boon JA, Ahlquist P. Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping. J Virol. 2000;74:8803–8811. - PMC - PubMed
    1. Alves-Rodrigues I, Galao RP, Meyerhans A, Diez J. Saccharomyces cerevisiae: A useful model host to study fundamental biology of viral replication. Virus Res. 2006;120:49–56. - PMC - PubMed
    1. Alves-Rodrigues I, Mas A, Diez J. Xenopus Xp54 and human RCK/p54 helicases functionally replace yeast Dhh1p in brome mosaic virus RNA replication. J Virol. 2007;81:4378–4380. - PMC - PubMed
    1. Barends S, Rudinger-Thirion J, Florentz C, Giege R, Pleij CW, Kraal B. tRNA-like structure regulates translation of brome mosaic virus RNA. J Virol. 2004;78:4003–4010. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources