[Polyadenylation of Sine Transcripts Generated by RNA Polymerase III Dramatically Prolongs Their Lifetime in Cells]

Area of Science:

• Molecular Biology
• Genetics
• RNA Biology

Background:

• Short Interspersed Elements (SINEs) are mobile genetic elements transcribed by RNA polymerase III.
• Some mammalian SINE transcripts can undergo unique AAUAAA-dependent polyadenylation.
• Polyadenylation of RNA polymerase II transcripts typically enhances cellular stability.

Purpose of the Study:

• To investigate if AAUAAA-dependent polyadenylation enhances the stability of SINE transcripts.
• To determine the impact of polyadenylation on SINE RNA half-life in HeLa cells.

Main Methods:

• Transfection of HeLa cells with SINE DNA (with and without polyadenylation signal).
• Inhibition of transcription using actinomycin D.
• Monitoring SINE transcript levels via northern hybridization.

Main Results:

• Nonpolyadenylated SINE transcripts had a half-life of 20-30 minutes.
• Polyadenylated SINE transcripts exhibited a half-life exceeding 3 hours.
• Structural modifications in B2 SINE did not significantly affect polyadenylated transcript stability.

Conclusions:

• AAUAAA-dependent polyadenylation dramatically increases SINE transcript stability.
• Poly(A) tail interaction with poly(A)-binding proteins (PABPs) likely protects SINEs from degradation.
• SINE-based vectors can be engineered for stable expression of short noncoding RNAs.