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

Abstract

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.

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

“…We previously demonstrated that recombinant 18S rRNA expressed from a plasmid in mouse N2a cells was correctly processed and incorporated into fully functional ribosomal subunits capable of mediating global protein synthesis as well as translation driven by poliovirus and encephalomyocarditis virus IRESs (29). As an initial step toward a functional analysis of the hypothesized basepairing interaction between HCV IRES and 18S rRNA, we examined whether our synthetic 18S rRNA expression system supports HCV IRES-dependent translation.…”

“…Cells were transfected with a plasmid that expresses mouse 18S rRNA with a G963A mutation (mouse numbering) that confers resistance to pactamycin ( Fig. S1A) (29). After 48 h, cells were subsequently transfected with a series of dicistronic reporter constructs, each encoding Renilla luciferase (Rluc) and Photinus luciferase (Pluc) from the first and second cistron, respectively ( Fig.…”

“…Constructs expressing mouse 18S rRNA (p18S series in this study) were based on p18S.1 (29). These constructs have a G-to-A point mutation at nt 963 (mouse numbering) that confers pactamycin resistance, and an insertion of a neutral hybridization tag for Northern blot analysis at the terminal loop of helix 44 (Fig.…”

“…Preparation of total RNA from transfected cells was performed essentially as described previously (41). Expression levels of the recombinant 18S rRNAs were determined by Northern analysis as described previously (29) with the exception of using a 32 P-labeled oligonucleotide probe (5′-GATATTCGGCAAGCAGGC-3′) that detected the hybridization tag sequence in helix 44 (Fig. S1A) or a probe (5′-CCAGGGCCGTGGGCCGAC-3′) that detected endogenous 18S rRNAs.…”

“…However, until recently, it has not been possible to directly test the predicted pairing interaction as it has not been possible to analyze mutated 18S rRNAs in mammalian cells. Here, we test the predicted basepairing interaction using a synthetic 18S rRNA expression system that we have developed in mouse cells (29). This system recapitulates the functionality of the native 18S rRNA, including the ability to support translation of exogenous genes.…”