The Biography of Ribonuclease P

Marta Wegorzewska

Macromolecules

5.7.09

www.physorg.com/news144947904.html

Discovery: pre-tRNA

Sidney Altman

Altman et al., 1971

1971: Precursor t-RNA

www.oisb.ca/.../pic_members_Sidney_Altman.jpg

Discovery:

Altman et al., 1971

41 nt at 5’ end

3 nt at 3’ end

Isolation and purification of pre-tRNA

Discovery:

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Evans et al., 2006

Altman et al., 1971

--------> Nuclear extracts of E.coli

RNase P

Discovery: purification of RNase P

Robertson et al., 1972

Purifying RNase P Actions of purified RNase P

1972:

Discovery: RNase P: RNA-protein mix

Stark et al., 1978

1978: Benjamin Stark (graduate student) identified the RNA and protein subunit of E.coli RNase P

M2: methylene blue staining for nucleic acid stain

C5: Coomassie brilliant blue staining for protein

Discovery: RNase P: a ribonuclease and ribozyme

6= E.coli RNA + protein

7= E.coli RNA (M1 RNA)

8= E.coli protein (C5)

Pre-tRNA

Mature tRNA

1983: Cecilia Guerrier-Takada

Guerrier-Takada et al., 1983

Conservation: RNA

Evans et al., 2006

Conservation: Protein

Hartmann et al., 2003Bacteria

Summary:

Bacteria Archaea Eukarya

RNA-

Protein-

E.coliM1 RNA; type A (ancestral) B. sub type B (Bacillus)

M. the. one RNA subunit

H. sap.

H1 RNA

E.coli one protein: C5 B. sub one protein: P protein

M. the. 4 proteins: Pop4, Rpr2, Rpp1, Pop5

H. sap.

10 proteins: hPop1, Rpp29, Rpp21, Rpp30, hPop5, Rpp14, Rpp20, Rpp25, Rpp40, Rpp38

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Hall et al., 2002

Function: ribonuclease

www.science.ca/images/altman_rnase.jpg

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Rnase P, Mg2+

Evans et al., 2006

RNase P functions to remove extraneous 5' sequences from precursor tRNAs to generate mature tRNA

Mechanism of Action:

1. Pre-tRNA binding to S and C domains of RNase P RNA subunit

2. RNase P protein subunit binding to 5’ end of pre-tRNA

Function: Human Rnase P: a transcription factor

Jarrous et al., 2007

RNase P protein found at 5S rRNA, 7SL RNA and U6 snRNA genes (non-RNase P substrate genes) and tRNA genes (RNase P substrate genes)

Potential as a tool: potential as a antibacterial, antiviral and anticancer agent

Can we take advantage of the catalytic function of M1RNA to target bacterial, viral, oncogenic mRNAs??

Cobaleda et al., 2001

Guide sequences (GS):

External guide sequences (EGS): exogenous GS recruits endogenous RNase P

Internal guide sequences (IGS): GS covalently linked to M1RNA (M1GS)Requirements:

1. Complementary to target mRNA

2. 3’ sequence for recognition by M1 RNA (EGS)

Cobaleda et al., 2001

Application: Anti-bacterial potential: Guerrier-Takada et al. 1995 showed specific targeting of EGS to B-galactosidase and alkaline phosphatase encoding genes (expressions were decreased by 50-60% in E.coli)

Anti-viral potential: IGS and EGS used to target herpes simplex virus 1 (HSV-1), human immunodeficiency virus (HIV), human influenza virus, human cytomegalovirus and Kaposi's sarcoma-associated herpesvirus

Cobaleda et al., 2001

Anti-cancer potential: M1GS used for destruction of chimeric mRNAs created by chromosomal translocation (BCR-ABL)

Application:Anti-cancer potential:

Ba/F3 cells expressing the human BCR-ABLp190

+

M1GS against BCR-ABL p190Cobaledo et al., 2000

Application: advantages for basic science research

This sounds awesome! Why don’t hear about it as a tool for used in gene knockdown studies?

Advantages of EGS/M1GS:

1. EGS uses endogenous RNase P (most abundant, stable and efficient enzymes) resulting in irreversible cleavage of target mRNA

2. Highly specific and does not mistarget (RNA i) 3. Little sign of cytotoxicity

RNA interference

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