Nuclease protection analysis of ribonucleoprotein complexes: use of the cytotoxic ribonuclease alpha-sarcin to determine the binding sites for Escherichia coli ribosomal proteins L5, L18, and L25 on 5S rRNA.

Huber, Paul W.; Wool, Ira G.

doi:10.1073/pnas.81.2.322

Cited by 63 publications

(16 citation statements)

References 32 publications

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“…Other previously studied examples of RNA structures that interact directly with protein include the RNAs which bind to bacteriophage R17 coat protein (4), ribosomal protein L54 (which binds 5S RNA [15,16]), and tRNA synthetases (14,31,39). In R17, a 21-nt RNA stem-loop was analyzed by saturation mutagenesis for the contribution of each nucleotide to the binding to R17 coat protein (4,38).…”

Section: Resultsmentioning

confidence: 99%

A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

1989

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We have defined the nucleotide sequence of a protein-binding domain within Ul RNA that specifically recognizes and binds both to a Ul small nuclear ribonucleoprotein component (the 70K protein) and to the previously defined RNA-binding domain of the 70K protein. We have investigated direct interactions between purified Ul RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of Ul RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of Ul RNA that are involved in base pairing with the 5' splice site of pre-mRNA were not required for binding. In contrast to other reports, these findings demonstrate that a specific domain of Ul RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.The 52-kilodalton Ul RNA-associated protein (the 70K protein) is a component of the Ul small nuclear ribonucleoprotein (snRNP) complex. Several snRNPs have been implicated in the removal of intervening sequences during precursor mRNA splicing (see references 11, 25, and 41 for reviews). The Ul snRNP, in particular, has been shown to interact with the 5' splice site, at least in part by base pairing of this site with the 5'-terminal 10 nucleotides (nt) of Ul RNA (1,5,18,29,45). The human Ul snRNP is composed of 165 nt of Ul RNA and two classes of proteins: Ul-specific proteins (70K, A, and C) and the Sm complex (consisting of six U snRNP-common proteins) (9, 22). Although these proteins probably play important structural roles in snRNPs and at least some are required for the interaction of Ul snRNP with 5' splice sites (29), none have been assigned specific functions.Previous studies of the RNA-binding properties of the Ul snRNP-specific proteins have involved indirect methods including immunoprecipitation of nuclease-treated cell extracts (34) and microinjection of RNA into Xenopus oocytes (12). These studies suggested that the 70K protein, as well as the A protein and perhaps the C protein, associates with the 5' half of Ul RNA, in particular, stem-loop I. In addition, weak requirements were implicated for regions of stemloops II, III, and IV (12,34 purified human Ul RNA and 70K protein, we have investigated the direct interaction of these two components of the Ul snRNP. Using various binding methods, we maintained the specificity of the Ul RNA-70K reconstitution through progressive deletion of both components. We previously defined the RNA-binding domain of the 70K protein required for this interaction (35). In this study, we report that both the 70K protein and the RNA-binding domain alone are directly recognized by 31 nt of Ul RNA that make up the 5'-most stem-loop structure. The A, C, and Sm proteins were not required for this recognition and binding. Further deletion of the 31-nt structure resulted in reduced efficiency of reconstitution, thus defining the minimal RNA domain needed for recognition of the 70K protein. MATERIALS AND METHODS

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Section: Resultsmentioning

confidence: 99%

A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

1989

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“…In order to localize MRP-L18-binding sites on human 5S rRNA, we measured dissociation constants of complexes between MRP-L18 and several mutated versions of human 5S rRNA (Table 1). These mutations, although not identical to those described in other studies, were created to disrupt, exchange, or delete the structural elements previously shown to be involved in L18 or eL5 binding in both structural (Ban et al 2000;Woestenenk et al 2002) and biochemical (Aoyama et al 1984;Huber and Wool 1984;Scripture and Huber 1995;Gongadze et al 2001) studies. Mutations in the b-domain and loop A-sites normally responsible for interaction of 5S rRNA with L18/eL5 proteins (Ban et al 2000)-did not significantly influence the binding.…”

Section: Mitochondrial Ribosomal Protein L18 Binds the Cytosolic 5s Rrnamentioning

confidence: 99%

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18

Smirnov

Entelis²,

Martin³

et al. 2011

Genes Dev.

106

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5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes.

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“…Deletion of a highly conserved A49-A50 two-nucleotide bulge in helix III as well as of loop C [D(35-42)], both needed for L18/eL5 family ribosomal proteins binding (see Fig. 1A; Huber and Wool 1984;Chow et al 1992;Scripture and Huber 1995;Huber et al 2001), led to no significant changes in 5S rRNA import efficiency. Even a more spacious deletion [D(28-51)], removing both helix III and loop C and destructing loop B structure, failed to substantially affect the import capacity of the 5S rRNA, in perfect agreement with the data presented above.…”

Section: Effects Of Deletions In the B-domain On The 5s Rrna Import Ementioning

confidence: 99%

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

Smirnov¹,

Tarassov²,

Mager-Heckel³

et al. 2008

RNA

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RNA import into mitochondria is a widespread phenomenon. Studied in details for yeast, protists, and plants, it still awaits thorough investigation for human cells, in which the nuclear DNA-encoded 5S rRNA is imported. Only the general requirements for this pathway have been described, whereas specific protein factors needed for 5S rRNA delivery into mitochondria and its structural determinants of import remain unknown. In this study, a systematic analysis of the possible role of human 5S rRNA structural elements in import was performed. Our experiments in vitro and in vivo show that two distinct regions of the human 5S rRNA molecule are needed for its mitochondrial targeting. One of them is located in the proximal part of the helix I and contains a conserved uncompensated G:U pair. The second and most important one is associated with the loop E-helix IV region with several noncanonical structural features. Destruction or even destabilization of these sites leads to a significant decrease of the 5S rRNA import efficiency. On the contrary, the b-domain of the 5S rRNA was proven to be dispensable for import, and thus it can be deleted or substituted without affecting the 5S rRNA importability. This finding was used to demonstrate that the 5S rRNA can function as a vector for delivering heterologous RNA sequences into human mitochondria. 5S rRNA-based vectors containing a substitution of a part of the b-domain by a foreign RNA sequence were shown to be much more efficiently imported in vivo than the wild-type 5S rRNA.

show abstract

Nuclease protection analysis of ribonucleoprotein complexes: use of the cytotoxic ribonuclease alpha-sarcin to determine the binding sites for Escherichia coli ribosomal proteins L5, L18, and L25 on 5S rRNA.

Cited by 63 publications

References 32 publications

A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

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