Eukaryotic RNase P

Mann, Hagit; Ben-Asouli, Yitzhak; Schein, Aleks; Moussa, Sana; Jarrous, Nayef

doi:10.1016/s1097-2765(03)00357-5

Cited by 72 publications

(39 citation statements)

References 44 publications

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“…M1 RNA may further increase its activity in cultured cells by interacting with cellular proteins (22,31). These properties, as well as the simple design of the guide sequence, make M1GS an attractive and unique gene-targeting agent that can be used generally for basic research and clinical applications.…”

Section: Discussion Engineered Rnase P Ribozyme As a Tool For Studiesmentioning

confidence: 99%

Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron cryomicroscopy

Trang²,

Shah³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Human CMV (HCMV) is the leading viral cause of birth defects and causes one of the most common opportunistic infections among transplant recipients and AIDS patients. Cleavage of internal scaffolding proteins by the viral protease (Pr) occurs during HCMV capsid assembly. To gain insight into the mechanism of HCMV capsid maturation and the roles of the Pr in viral replication, an RNase P ribozyme was engineered to target the Pr mRNA and down-regulate its expression by >99%, generating premature Pr-minus capsids. Furthermore, scaffolding protein processing and DNA encapsidation were inhibited by 99%, and viral growth was reduced by 10,000-fold. 3D structural comparison of the Pr-minus and wild-type B capsids by electron cryomicroscopy, at an unprecedented 12.5-Å resolution, unexpectedly revealed that the structures are identical in their overall shape and organization. However, the Pr-minus capsid contains tenuous connections between the scaffold and the capsid shell, whereas the wild-type B capsid has extra densities in its core that may represent the viral Pr. Our findings indicate that cleavage of the scaffolding protein is not associated with the morphological changes that occur during capsid maturation. Instead, the protease appears to be required for DNA encapsidation and the subsequent maturation steps leading to infectious progeny. These results therefore provide key insights into an essential step of HCMV infection using an RNase P ribozyme-based inhibition strategy.RNase P ͉ herpesvirus ͉ antisense ͉ catalytic RNA ͉ structure H uman CMV (HCMV) is a member of the human herpesvirus family, which also includes herpes simplex virus 1 (HSV-1) and HSV-2, varicella-zoster virus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus (1-4). The double-stranded DNA genome is encapsidated by an icosahedral protein capsid, which is surrounded by an amorphous layer of proteins called tegument. The entire particle is enclosed in a lipid bilayer envelope containing viral glycoproteins. The capsid is assembled initially with the formation of an internal protein scaffold, followed by proteolytic cleavage and removal of the scaffold and packaging of viral genome in the core (1-4).HCMV is the leading viral cause of birth abnormalities (5) and causes one of the most common opportunistic infections encountered in AIDS patients and organ transplant recipients (6). Like other herpesviruses, HCMV virions contain multiple layers whose assembly requires several steps. In HCMV, UL80.5, which encodes preassembly͞scaffold protein (preAP), overlaps with the 3Ј coding sequence of UL80 that codes for the protease (Pr). The UL80 product processes itself internally to release an N-terminal fragment, proteolytic protein (Ϸ30 kDa), NP1n, and a C-terminal fragment, NP1c protein, which overlaps with preAP (7). The Pr also cleaves preAP at its C-terminal region, which is shown to interact with the capsid shell (8), although this interaction has not been well defined in HCMV. For all herpesviruses, four intermediate capsid types are ...

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Section: Discussion Engineered Rnase P Ribozyme As a Tool For Studiesmentioning

confidence: 99%

Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron cryomicroscopy

Trang²,

Shah³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Rpp21, Rpp29 and H1 RNA are sufficient for reconstitution of RNase P activity in tRNA processing in vitro (24). The archaeal Rpp21, Rpp29, Pop5, Rpp30 and Rpp38 proteins are required for efficient reconstitution of thermostable RNase P ribonucleoprotein (23,25–27).…”

Section: Nuclear Rnase P: An Ensemble Of Rna With Highly Conserved Prmentioning

confidence: 99%

“…Recent progress in modeling the tertiary folding of eukaryal RNase P RNA uncovers that it has a conserved core structure similar to that of its bacterial counterpart (29). Mutations that disrupt the predicted tertiary folding of H1 RNA abolish catalysis in vitro (24). Remarkably, Kikovska et al, has shown that H1 RNA is active in tRNA processing in vitro in the absence of any protein (30), a finding that is consistent with previous observation that H1 RNA alone binds to precursor tRNAs in vitro and has a conserved catalytic core (24,29).…”

Section: Nuclear Rnase P: An Ensemble Of Rna With Highly Conserved Prmentioning

confidence: 99%

Human RNase P: a tRNA-processing enzyme and transcription factor

Jarrous

Reiner

2007

Nucleic Acids Research

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Ribonuclease P (RNase P) has been hitherto well known as a catalytic ribonucleoprotein that processes the 5′ leader sequence of precursor tRNA. Recent studies, however, reveal a new role for nuclear forms of RNase P in the transcription of tRNA genes by RNA polymerase (pol) III, thus linking transcription with processing in the regulation of tRNA gene expression. However, RNase P is also essential for the transcription of other small noncoding RNA genes, whose precursor transcripts are not recognized as substrates for this holoenzyme. Accordingly, RNase P can act solely as a transcription factor for pol III, a role that seems to be conserved in eukarya.

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“…Reconstitution using all or some recombinant components has been recently reported for archaeal [Pyrococcus horikoshii (Pho) and Mth] and eukaryal (human) RNase P (22)(23)(24). In these cases, however, the reconstituted RNase P holoenzymes displayed weak activity (compared with either their respective native versions or holoenzymes from other species) and were not fully characterized.…”

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confidence: 99%

“…It remains to be proven whether the partial (functional) assemblages observed in Pfu RNase P represent common intermediates during hierarchical assembly of archaeal͞eukaryal RNase P holoenzymes. In this regard, it is notable that weak activity was observed when human RPR was reconstituted with human Rpp21 and Rpp29 (24).…”

mentioning

confidence: 99%

Functional reconstitution and characterization of Pyrococcus furiosus RNase P

Tsai

Pulukkunat

Woznick

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

202

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RNase P, which catalyzes the magnesium-dependent 5-end maturation of tRNAs in all three domains of life, is composed of one essential RNA and a varying number of protein subunits depending on the source: at least one in bacteria, four in archaea, and nine in eukarya. To address why multiple protein subunits are needed for archaeal͞eukaryal RNase P catalysis, in contrast to their bacterial relative, in vitro reconstitution of these holoenzymes is a prerequisite. Using recombinant subunits, we have reconstituted in vitro the RNase P holoenzyme from the thermophilic archaeon Pyroccocus furiosus (Pfu) and furthered our understanding regarding its functional organization and assembly pathway(s). Whereas Pfu RNase P RNA (RPR) alone is capable of multiple turnover, addition of all four RNase P protein (Rpp) subunits to Pfu RPR results in a 25-fold increase in its k cat and a 170-fold decrease in Km. In fact, even in the presence of only one of two specific pairs of Rpps, the RPR displays activity at lower substrate and magnesium concentrations. Moreover, a pared-down, mini-Pfu RNase P was identified with an RPR deletion mutant. Results from our kinetic and footprinting studies on Pfu RNase P, together with insights from recent structures of bacterial RPRs, provide a framework for appreciating the role of multiple Rpps in archaeal RNase P.archaeal RNase P ͉ in vitro reconstitution ͉ precursor tRNA processing R ibonuclease P (RNase P) is an ancient and essential endoribonuclease that catalyzes the 5Ј-end maturation of tRNAs in all three domains of life (1-7). Whereas RNase P in all living organisms contains one essential RNA subunit, the number of protein cofactors͞subunits varies: at least one in bacterial (8), four in archaeal (9), and nine in eukaryal (nuclear) RNase P (10, 11). The basis for this variation, which has implications for macromolecular evolution, is unclear.The bacterial RNase P RNA (RPR) alone is catalytically active under in vitro conditions of high ionic strength in the presence of a divalent ion such as Mg 2ϩ (1); however, the protein cofactor is essential for RNase P function in vivo because of its pleiotropic effects on RNA structure, substrate recognition, affinity for Mg 2ϩ , and precursor tRNA (ptRNA) cleavage (12-19). Although phylogenetic sequence analysis revealed that archaeal and eukaryal RPRs likely retain the same catalytic core as the bacterial ribozyme, many archaeal and all eukaryal RPRs seem to be incapable of supporting catalysis in the absence of their protein cofactors (6,7,20,21). Genetic and biochemical studies established the association of yeast͞human nuclear RNase P activity with at least nine protein subunits (seven of which are homologs) (10, 11). RNase P activity could be immunoprecipitated from a partially purified Methanothermobacter thermoautotrophicus (Mth) RNase P preparation by using polyclonal antisera generated against four Mth polypeptides that exhibit sequence homology to four of the seven conserved yeast͞human RNase P proteins (Rpps; ref. 9). None of the archaeal...

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Eukaryotic RNase P

Cited by 72 publications

References 44 publications

Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron cryomicroscopy

Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron cryomicroscopy

Human RNase P: a tRNA-processing enzyme and transcription factor

Functional reconstitution and characterization of Pyrococcus furiosus RNase P

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