1. The Biography of Ribonuclease P
Marta Wegorzewska
Macromolecules
5.7.09

2. Discovery: pre-tRNA 1971: Precursor t-RNA Sidney Altman
Altman et al., 1971

3. Discovery: 41 nt at 5’ end 3 nt at 3’ end
Isolation and purification of
pre-tRNA
Altman et al., 1971

4. Discovery: -----------------------------------------> RNase P
Evans et al., 2006
Nuclear extracts of E.coli
>
Altman et al., 1971

5. Discovery: purification of RNase P
1972:
Purifying RNase P Actions of purified RNase P
Robertson et al., 1972

6. Discovery: RNase P: RNA-protein mix
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
Stark et al., 1978

7. Discovery: RNase P: a ribonuclease and ribozyme
1983: Cecilia Guerrier-Takada
6= E.coli RNA + protein
7= E.coli RNA (M1 RNA)
8= E.coli protein (C5)
Pre-tRNA
Mature tRNA
Guerrier-Takada et al., 1983

8. Conservation: RNA
Evans et al., 2006

9. Conservation: Protein
Eukarya
Archaea
Bacteria
Hartmann et al., 2003

10. Summary: Bacteria Archaea Eukarya RNA- Protein- E.coli
M1 RNA; type A (ancestral)
B. sub type B (Bacillus)
RNA-
Protein-
M. the one RNA subunit
H. sap.
H1 RNA
H. sap.
10 proteins: hPop1, Rpp29, Rpp21, Rpp30, hPop5, Rpp14, Rpp20, Rpp25, Rpp40, Rpp38
E.coli one protein: C B. sub one protein: P protein
M. the.
4 proteins:
Pop4, Rpr2,
Rpp1, Pop5
Hall et al., 2002

11. Function: ribonuclease
RNase P functions to remove extraneous
5' sequences from precursor tRNAs
to generate mature tRNA
Rnase P, Mg2+
Evans et al., 2006

12. Mechanism of Action:
Pre-tRNA binding to S and C domains of RNase P RNA subunit
RNase P protein subunit binding to 5’ end of pre-tRNA

13. Function: Human Rnase P: a transcription factor
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)
Jarrous et al., 2007

14. Potential as a tool: potential as a antibacterial, antiviral and anticancer agent
Cobaleda et al., 2001
Can we take advantage of the catalytic function of M1RNA to target bacterial, viral, oncogenic mRNAs??

15. Guide sequences (GS):
External guide sequences (EGS): exogenous GS recruits endogenous RNase P
Internal guide sequences (IGS): GS covalently linked to M1RNA (M1GS)
Requirements:
Complementary to target mRNA
3’ sequence for recognition by M1 RNA (EGS)
Cobaleda et al., 2001

16. Application:
Anti-bacterial potential: Guerrier-Takada et al 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
Anti-cancer potential: M1GS used for destruction of chimeric mRNAs created by chromosomal translocation (BCR-ABL)
Cobaleda et al., 2001

17. Application: + Anti-cancer potential: M1GS against BCR-ABL p190
Ba/F3 cells expressing the human BCR-ABLp190 +
M1GS against BCR-ABL p190
Cobaledo et al., 2000

18. Application: advantages for basic science research
This sounds awesome! Why don’t hear about it as a tool for used in gene knockdown studies?
RNA interference
Advantages of EGS/M1GS:
EGS uses endogenous RNase P (most abundant, stable and efficient enzymes) resulting in irreversible cleavage of target mRNA
Highly specific and does not mistarget (RNA i)
Little sign of cytotoxicity