Nuclease activity associated with mammalian mRNA in its native state: possible basis for selectivity in mRNA decay

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Mammalian cell extracts, including polysome and messenger ribonucleoprotein (mRNP) preparations, possess inherent nuclease activity.
• This nuclease activity degrades messenger RNA (mRNA) within these preparations, complicating molecular studies.

Purpose of the Study:

• To characterize the nuclease activity associated with mammalian polysomes and mRNPs.
• To investigate the substrate specificity and cleavage patterns of these mRNA-degrading enzymes.

Main Methods:

• Cosedimentation analysis of nuclease activity with polysomes and mRNPs.
• EDTA treatment to release mRNPs and assess nuclease association.
• Incubation of purified beta-globin mRNA and various endogenous mRNAs (P40, P21, actin) with different polysome preparations to determine cleavage sites.

Main Results:

• Nuclease activity cosediments with polysomes and free mRNPs, remaining associated after EDTA treatment, but not with ribosomal subunits.
• mRNA degradation rates are unaffected by dilution, indicating tight enzyme-substrate association.
• Specific cleavage sites were identified on beta-globin mRNA (AU sequences), P40 mRNA (3' noncoding region), P21 mRNA (5' noncoding region), and actin mRNA (internal region).
• Mouse sarcoma 180 (S-180) polysomes induced additional cleavages at UG sequences on beta-globin mRNA.

Conclusions:

• Mammalian cells contain mRNA-bound nucleases tightly associated with mRNPs.
• These nucleases exhibit specific cleavage patterns, suggesting targeted interactions with individual mRNA species.
• The findings provide insights into mRNA regulation and degradation mechanisms within the cell.