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Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae.
RNA. 2013 Dec; 19(12):1639-47.RNA

Abstract

Eukaryotic ribosome assembly requires over 200 assembly factors that facilitate rRNA folding, ribosomal protein binding, and pre-rRNA processing. One such factor is Rlp7, an essential RNA binding protein required for consecutive pre-rRNA processing steps for assembly of yeast 60S ribosomal subunits: exonucleolytic processing of 27SA3 pre-rRNA to generate the 5' end of 5.8S rRNA and endonucleolytic cleavage of the 27SB pre-rRNA to initiate removal of internal transcribed spacer 2 (ITS2). To better understand the functions of Rlp7 in 27S pre-rRNA processing steps, we identified where it crosslinks to pre-rRNA. We found that Rlp7 binds at the junction of ITS2 and the ITS2-proximal stem, between the 3' end of 5.8S rRNA and the 5' end of 25S rRNA. Consistent with Rlp7 binding to this neighborhood during assembly, two-hybrid and affinity copurification assays showed that Rlp7 interacts with other assembly factors that bind to or near ITS2 and the proximal stem. We used in vivo RNA structure probing to demonstrate that the proximal stem forms prior to Rlp7 binding and that Rlp7 binding induces RNA conformational changes in ITS2 that may chaperone rRNA folding and regulate 27S pre-rRNA processing. Our findings contradict the hypothesis that Rlp7 functions as a placeholder for ribosomal protein L7, from which Rlp7 is thought to have evolved in yeast. The binding site of Rlp7 is within eukaryotic-specific RNA elements, which are not found in bacteria. Thus, we propose that Rlp7 coevolved with these RNA elements to facilitate eukaryotic-specific functions in ribosome assembly and pre-rRNA processing.

Authors

No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

24129494

Citation

Dembowski, Jill A., et al. "Identification of the Binding Site of Rlp7 On Assembling 60S Ribosomal Subunits in Saccharomyces Cerevisiae." RNA (New York, N.Y.), vol. 19, no. 12, 2013, pp. 1639-47.
Dembowski JA, Ramesh M, McManus CJ, et al. Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae. RNA. 2013;19(12):1639-47.
Dembowski, J. A., Ramesh, M., McManus, C. J., & Woolford, J. L. (2013). Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae. RNA (New York, N.Y.), 19(12), 1639-47. https://doi.org/10.1261/rna.041194.113
Dembowski JA, et al. Identification of the Binding Site of Rlp7 On Assembling 60S Ribosomal Subunits in Saccharomyces Cerevisiae. RNA. 2013;19(12):1639-47. PubMed PMID: 24129494.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae. AU - Dembowski,Jill A, AU - Ramesh,Madhumitha, AU - McManus,C Joel, AU - Woolford,John L,Jr Y1 - 2013/10/15/ PY - 2013/10/17/entrez PY - 2013/10/17/pubmed PY - 2014/1/18/medline KW - RNA binding protein KW - Rlp7 KW - crosslinking and analysis of cDNAs (CRAC) KW - ribosomal protein L7 KW - ribosome biogenesis SP - 1639 EP - 47 JF - RNA (New York, N.Y.) JO - RNA VL - 19 IS - 12 N2 - Eukaryotic ribosome assembly requires over 200 assembly factors that facilitate rRNA folding, ribosomal protein binding, and pre-rRNA processing. One such factor is Rlp7, an essential RNA binding protein required for consecutive pre-rRNA processing steps for assembly of yeast 60S ribosomal subunits: exonucleolytic processing of 27SA3 pre-rRNA to generate the 5' end of 5.8S rRNA and endonucleolytic cleavage of the 27SB pre-rRNA to initiate removal of internal transcribed spacer 2 (ITS2). To better understand the functions of Rlp7 in 27S pre-rRNA processing steps, we identified where it crosslinks to pre-rRNA. We found that Rlp7 binds at the junction of ITS2 and the ITS2-proximal stem, between the 3' end of 5.8S rRNA and the 5' end of 25S rRNA. Consistent with Rlp7 binding to this neighborhood during assembly, two-hybrid and affinity copurification assays showed that Rlp7 interacts with other assembly factors that bind to or near ITS2 and the proximal stem. We used in vivo RNA structure probing to demonstrate that the proximal stem forms prior to Rlp7 binding and that Rlp7 binding induces RNA conformational changes in ITS2 that may chaperone rRNA folding and regulate 27S pre-rRNA processing. Our findings contradict the hypothesis that Rlp7 functions as a placeholder for ribosomal protein L7, from which Rlp7 is thought to have evolved in yeast. The binding site of Rlp7 is within eukaryotic-specific RNA elements, which are not found in bacteria. Thus, we propose that Rlp7 coevolved with these RNA elements to facilitate eukaryotic-specific functions in ribosome assembly and pre-rRNA processing. SN - 1469-9001 UR - https://www.unboundmedicine.com/medline/citation/24129494/Identification_of_the_binding_site_of_Rlp7_on_assembling_60S_ribosomal_subunits_in_Saccharomyces_cerevisiae_ L2 - http://www.rnajournal.org/cgi/pmidlookup?view=long&pmid=24129494 DB - PRIME DP - Unbound Medicine ER -