Molecular Modeling of the Three-dimensional Structure of the Bacterial RNase P Holoenzyme

Tsai, Hui Lien; Masquida, Benoı̂t; Biswas, Rajarshi; Westhof, Éric; Gopalan, Venkat

doi:10.1016/s0022-2836(02)01267-6

Cited by 107 publications

(119 citation statements)

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“…Sequences M1 RNA interacts with C5 protein in the RNase P holoenzyme, whereas the P8/9 and P12 regions are not essential for the interaction [30,31]. We assessed binding of these truncated M1 RNA derivatives to C5 protein.…”

Section: Namesmentioning

confidence: 99%

Novel function of C5 protein as a metabolic stabilizer of M1 RNA

Kim

Lee

2008

FEBS Letters

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Edited by Ulrike KutayKeywords: M1 RNA C5 protein RNase P RNA stability a b s t r a c t Escherichia coli RNase P is a ribonucleoprotein composed of a large RNA subunit (M1 RNA) and a small protein subunit (C5 protein). We examined if C5 protein plays a role in maintaining metabolic stability of M1 RNA. The sequestration of C5 protein available for M1 RNA binding reduced M1 RNA stability in vivo, and its reduced stability was recovered via overexpression of C5 protein. In addition, M1 RNA was rapidly degraded in a temperature-sensitive C5 protein mutant strain at non-permissive temperatures. Collectively, our results demonstrate that the C5 protein metabolically stabilizes M1 RNA in the cell.

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Section: Namesmentioning

confidence: 99%

Novel function of C5 protein as a metabolic stabilizer of M1 RNA

Kim

Lee

2008

FEBS Letters

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“…RNA samples were isolated from parental U373MG cells (Ϫ, lanes 1, 6, 7, 12, 13, 18, 19, and 24) and cell lines that expressed R27-IE (lanes 2, 8, 14, and 20), V188-IE (lanes 3, 10, 15, and 21), C-IE (lanes 4, 10, 16, and 22), and M1-IE (lanes 5, 11, 17, and 23). These samples (30 g) were then separated on agarose gels that contained formaldehyde, transferred to a nitrocellulose membrane, and hybridized to 32 P-radiolabeled probes that contained the cDNA sequence of the HCMV 5-kb transcript (lanes 1-6), IE1 mRNA (lanes 7-12), IE2 mRNA (lanes [13][14][15][16][17][18], and US2 mRNA (lanes 19 -24). The hybridized products corresponding to the 5-kb RNA, IE1, IE2, and US2 mRNAs were about 5, 1.9, 2.2, and 1.5 kb, respectively (1,28,33).…”

Section: Engineered Rnase P Ribozyme As Antiviral Agentsribozyme Varimentioning

confidence: 99%

Engineered RNase P Ribozymes Are Efficient in Cleaving a Human Cytomegalovirus mRNA in Vitro and Are Effective in Inhibiting Viral Gene Expression and Growth in Human Cells

Zou

Lee

Umamoto

et al. 2003

Journal of Biological Chemistry

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By using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a ribozyme variant was used to target the overlapping region of the mRNAs encoding human cytomegalovirus (HCMV) major transcription regulatory proteins IE1 and IE2. The variant is about 90 times more efficient in cleaving the IE1/IE2 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Our results provide the first direct evidence that a point mutation at nucleotide position 80 of RNase P catalytic RNA from Escherichia coli (U 80 3 C 80 ) increases the rate of chemical cleavage, and another mutation at nucleotide position 188 (C 188 3 U 188 ) enhances substrate binding of the ribozyme. Moreover, the variant is more effective in inhibiting viral IE1 and IE2 expression and growth in HCMV-infected cells than the wild type ribozyme. A reduction of about 99% in the expression level of IE1 and IE2 and a reduction of 10,000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of less than 10% in IE1/IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered RNase P ribozyme variants are highly effective in inhibiting HCMV gene expression and growth. These results also demonstrate the feasibility of engineering highly effective RNase P ribozymes for gene targeting applications, including anti-HCMV gene therapy. Human cytomegalovirus (HCMV)1 causes significant morbidity and mortality in immunocompromised or immunologically immature individuals, including neonates and AIDS patients (1, 2). The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and novel treatment strategies. RNA enzymes are being developed as promising gene-targeting agents to specifically cleave RNA sequences of choice (3, 4). These ribozymes contain both a catalytic RNA domain that cleaves the target mRNA and a substrate-binding domain with a sequence antisense to the target mRNA sequence. Therefore, these gene-targeting ribozymes bind to the mRNA sequence through Watson-Crick interactions between the target sequence and the antisense sequence in the substrate-binding domain of the ribozyme. Compared with conventional antisense DNA and RNA, a ribozyme may have several unique features as it can cleave its target irreversibly, and multiple copies of its substrate can be cleaved by a single ribozyme molecule. Both hammerhead and hairpin ribozymes have been shown to cleave viral mRNA sequences and inhibit viral replication in cells infected with human viruses, whereas a ribozyme derived from a group I intron has been used to repair mutant mRNAs in cells (5-8). Thus, ribozymes can be used as a tool in both basic research and clinical applications, such as in studies of developmental processes and in antiviral gene therapy (3, 4).RNase P is a ribonucleoprotein complex responsib...

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“…Therefore, there is a crucial need for novel methods of determining the 3D structures of RNAs. Computational modeling of RNA 3D structures offers the opportunity to incorporate the structural features of RNAs extracted from known RNA structures (Das and Baker, 2007;Jonikas et al, 2009;Jossinet and Westhof, 2005;Major et al, 1993;Major et al, 1991;Massire et al, 1998;Parisien and Major, 2008;Shapiro et al, 2007;Tsai et al, 2003), to integrate physical and chemical principles (Cao and Chen, 2011;Ding et al, 2008), and to include experimentally derived structural information in modeling (Jonikas et al, 2009). For instance, several recent RNA 3D structure modeling methods (Cao and Chen, 2011;Das and Baker, 2007;Ding et al, 2008;Parisien and Major, 2008) have yielded accurate structure predictions of small RNAs from sequence alone, highlighting the predictive power of RNA modeling approaches in general.…”

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confidence: 99%