Terminal modification, sequence, length, and PIWI-protein identity determine piRNA stability

Area of Science:

• Molecular Biology
• RNA Biology
• Epigenetics

Background:

• PIWI-interacting RNAs (piRNAs) are essential for silencing transposons, combating viral infections, and regulating gene expression in animals.
• piRNA biogenesis involves critical 3' terminal modifications: trimming and 2'-O-methylation, which are known to influence piRNA stability.

Purpose of the Study:

• To investigate the roles of 3' terminal trimming and 2'-O-methylation in piRNA stability.
• To elucidate the mechanisms by which unmethylated piRNAs are destabilized.
• To understand how these modifications protect piRNAs from degradation pathways.

Main Methods:

• Biochemical assays were used to analyze piRNA stability in mouse and fly systems.
• Investigated complementarity-dependent destabilization mechanisms.
• Examined the impact of trigger RNA complementarity on piRNA degradation.
• Assessed the protective effects of 2'-O-methylation and trimming against specific degradation pathways.
• Analyzed the influence of uracil- and guanine-rich tracts on pre-piRNA decay in testis lysate and in vivo.

Main Results:

• Unmethylated mouse piRNAs are destabilized through multiple mechanisms, contingent on complementarity to trigger RNAs.
• 3' terminal 2'-O-methylation blocks complementarity-dependent destabilization of piRNAs in mice and flies, independent of extensive base pairing.
• 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent degradation in flies.
• Pre-piRNA trimming protects mouse piRNAs from a degradation pathway not dependent on trigger complementarity.
• Uracil- or guanine-rich tracts accelerate pre-piRNA decay in testis lysate and in vivo.
• Loss of both trimming and 2'-O-methylation leads to the collapse of the mouse piRNA pathway.

Conclusions:

• 3' terminal trimming and 2'-O-methylation are crucial collaborative modifications that stabilize piRNAs.
• These modifications provide distinct protective mechanisms against different degradation pathways, ensuring piRNA integrity.
• The combined action of trimming and methylation is essential for the proper functioning of the piRNA pathway.