Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P

Abstract

The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited.external guide sequences ͉ upstream sequences ͉ downstream sequences ͉ Rpp38T he regulation of expression of single genes in human cells by external manipulation is an important problem. RNA interference, the use of antisense RNA, and similar methods (1, 2) have been used with moderate success on cells in tissue culture. One other method, which uses RNase P as the agent that cleaves targeted RNAs, is also quite promising (3, 4). In the past, this mechanism has been used, for example, to decrease the amount of viral infection by regulating certain essential viral genes (5). This method, which uses external guide sequences (EGSs), has now been used to regulate expression of a gene that encodes one of the protein subunits of RNase P.Human nuclear RNase P has at least 10 protein subunits and one RNA subunit and is localized in the nuclei of cells (6-8). While using EGS action to study the expression of one of the subunits, Rpp38, we did not know whether any of the other proteins would also be affected. In fact, the mechanism for the control of several genes simultaneously in human cells is a diverse and puzzling problem. After surveying cells after transient transfection with a plasmid that harbored EGS Rpp38 by Western and Northern blots, we found an expected decrease in both the mRNA and translation product of Rpp38. As controls, we also explored the amounts of mRNA and protein made by other subunits of RNase P. Four other genes were inhibited, whereas the rest were unchanged. This finding led to an examination of what factors in terms of upstream and downstream DNA and RNA sequences were involved with this phenomenon and whether there could be a common regulatory feature. Materials and MethodsConstruction of EGSs and Plasmids. A DNA fragment that contains the Rpp38 cDNA under control of a T7 promoter was obtained by PCR using the full-length Rpp38 cDNA clone F3053 in PCR-Script-Amp (kindly provided by C. C. Liew, University of Toronto, Toronto) as a template. Primer oligonucleotides were P38F5 5Ј-TAA TAC GAC TCA CTA TAG GGC TCA AGT TATC-3Ј and P38BHIR ...

Regulated expression of functional external guide sequences in mammalian cells using a U6 RNA polymerase III promoter

“…Rpp14, Rpp20, Rpp30, and Rpp40 did not change significantly. These results are in agreement with our data obtained for HeLa cells, which expressed EGS Rpp38 after transient transfection (Kovrigina et al 2003). .…”

“…1A, bottom) and H1 RNA (data not shown) did not change during those 10 d of cell growth in the presence of ponasterone A. Inducible expression of EGS Rpp38 RNA in tissue culture decreases the amount of target Rpp38 mRNA and protein specifically (see below) and does not influence 5S and H1 RNA (data not shown). This last result with H1 RNA replicates an earlier finding on this system in transfection experiments (Kovrigina et al 2003).…”

“…The nature of this coordinate inhibition of gene expression is not yet understood. EGS Rpp38 was expressed transiently from a plasmid and its production was unregulated in all of those experiments (Kovrigina et al 2003). We also showed that lamin A/C was also decreased in gene expression by the expression of EGS Rpp38 , another aspect of down-regulation that remains to be understood.…”

“…Earlier, we designed an EGS that binds to the mRNA of Rpp38, one of the RNase P protein subunits, and forms a complex resembling the structure of a tRNA, and directs human RNase P to cleave the target mRNA (Kovrigina et al 2003). This EGS (EGS Rpp38 ) showed exquisite specificity and directed the RNase P-dependent cleavage at the targeted location within Rpp38 mRNA.…”

Ribonuclease P

“…The problem of getting the EGS into different cell types in animals is not trivial. Liposomes that envelop EGSs have been delivered to tissue culture cells with some success [20] but basic peptides covalently attached to morpholino oligonucleotides (PPMO; table 1) have been used recently with very good practical results [21]. The structure shown in figure 10, provided by AVI BioPharma (Bothell, WA, USA), has enabled the transfection of bacteria to change the phenotype of certain cell types.…”