Analysis of rRNA processing and translation in mammalian cells using a synthetic 18S rRNA expression system.

Nucleic Acids Res 2012 Sep 20;40(16):8085-98. Epub 2012 Jun 20.

Department of Neurobiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Analysis of processing, assembly, and function of higher eukaryotic ribosomal RNA (rRNA) has been hindered by the lack of an expression system that enables rRNA to be modified and then examined functionally. Given the potential usefulness of such a system, we have developed one for mammalian 18S rRNA. We inserted a sequence tag into expansion segment 3 of mouse 18S rRNA to monitor expression and cleavage by hybridization. Mutations were identified that confer resistance to pactamycin, allowing functional analysis of 40S ribosomal subunits containing synthetic 18S rRNAs by selectively blocking translation from endogenous (pactamycin-sensitive) subunits. rRNA constructs were suitably expressed in transfected cells, shown to process correctly, incorporate into ≈ 15% of 40S subunits, and function normally based on various criteria. After rigorous analysis, the system was used to investigate the importance of sequences that flank 18S rRNA in precursor transcripts. Although deletion analysis supported the requirement of binding sites for the U3 snoRNA, it showed that a large segment of the 5' external transcribed spacer and the entire first internal transcribed spacer, both of which flank 18S rRNA, are not required. The success of this approach opens the possibility of functional analyses of ribosomes, with applications in basic research and synthetic biology.

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gks530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3439915PMC
September 2012
4 Reads

Publication Analysis

Top Keywords

18s rrna
20
synthetic 18s
8
rrna
8
expression system
8
flank 18s
8
transcribed spacer
8
18s
6
analysis
5
constructs suitably
4
rrna constructs
4
expressed transfected
4
transfected cells
4
suitably expressed
4
cells process
4
40s subunits
4
subunits function
4
function based
4
based criteria
4
15% 40s
4
≈ 15%
4

Similar Publications