The effects of RNA degradation enzymes on antisense RNAI controlling ColE2 plasmid copy number

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• ColE2 plasmid replication is regulated by the Rep initiator protein.
• Rep expression is controlled by antisense RNA (RNAI), which inhibits Rep mRNA translation.
• Understanding RNAI degradation is crucial for plasmid stability.

Purpose of the Study:

• To investigate the effects of RNA degradation enzymes on the degradation pathways of ColE2 plasmid RNAI.
• To elucidate the roles of specific enzymes like RNase E, RNase II, PNPase, and PAP I in RNAI processing.
• To compare the RNAI degradation pathway with other related RNAs and the Rep mRNA.

Main Methods:

• Analysis of RNAI degradation intermediates in wild-type (wt) and DeltapcnB strains (lacking PAP I).
• Enzymatic assays to determine the cleavage sites and degradation kinetics of RNAI.
• Comparison of degradation pathways across different plasmid systems (ColE2, R1, ColE1).

Main Results:

• The RNAI degradation intermediate (RNAI(*)), generated by RNase E cleavage, accumulates significantly in the DeltapcnB strain.
• RNase II and PNPase are involved in the subsequent degradation of RNAI(*).
• Poly(A) polymerase I (PAP I) is essential for efficient RNAI degradation.
• ColE2 RNAI degradation shares similarities with R1 CopA RNA and ColE1 RNAI despite sequence differences.
• ColE2 RNAI is cleaved by RNase E at multiple 5' end positions.
• The RNAI degradation pathway differs from that of ColE2 Rep mRNA.
• The DeltapcnB strain exhibits a reduced ColE2 plasmid copy number.

Conclusions:

• ColE2 RNAI degradation is a multi-step process involving RNase E, RNase II, PNPase, and PAP I.
• PAP I plays a critical role in the efficient turnover of ColE2 RNAI.
• The identified degradation pathway provides insights into plasmid copy number control.
• Despite sequence variations, conserved mechanisms govern RNAI degradation in related plasmids.