TENT2, TUT4, and TUT7 selectively regulate miRNA sequence and abundance

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Terminal uridylate/adenylyl transferases (TENTs) are known to generate microRNA (miRNA) isoforms via 3' tailing.
• The precise mechanisms and functional consequences of TENT-mediated miRNA tailing remain largely uncharacterized.

Purpose of the Study:

• To elucidate the specific roles of TENT2, TUT4, and TUT7 in miRNA 3' tailing and subsequent functional regulation.
• To investigate the impact of different tailing modifications (adenylation, uridylation, guanylation) on miRNA stability and activity.

Main Methods:

• Generation of isogenic HEK293T cell lines with individual or combined knockouts of TENT2, TUT4, and TUT7.
• Deep sequencing, Northern blot analysis, and in vitro assays to characterize TENT-specific effects on miRNA isoforms.
• Rescue experiments to validate findings from knockout studies.

Main Results:

• 3' tailing of miRNAs is a highly specific process, not random.
• TENT2 exhibits adenylation, guanylation, and uridylation activity on mature miRNAs.
• TUT4 is responsible for the uridylation of most miRNAs, while TUT7 appears dispensable for this process.
• Abolishing uridylation significantly dysregulates specific miRNAs, whereas removing adenylation has a minor effect.
• Distinct regulatory mechanisms by TUT4/7 impact miR-181b and miR-222, while TUT7 specifically upregulates the miR-888 cluster.

Conclusions:

• TENT enzymes play selective roles in generating 3' miRNA isomiRs.
• Uridylation is a critical modification for the regulation of a subset of miRNAs.
• These findings provide a foundation for exploring the functional significance of TENT-mediated miRNA modifications.