Messenger RNAs that are not synthesized by RNA polymerase II can be 3' end cleaved and polyadenylated

Area of Science:

• Molecular Biology
• Virology
• Gene Expression

Background:

• Influenza virus mRNA polyadenylation relies on viral RNA polymerase copying a U5-7 sequence.
• Cellular polyadenylation of eukaryotic pre-mRNAs is generally considered coupled to RNA polymerase II (pol II) transcription.
• This coupling suggests that only pol II-synthesized RNAs are efficiently polyadenylated by the cellular machinery.

Purpose of the Study:

• To investigate whether the cellular polyadenylation machinery can process RNA transcripts not synthesized by RNA polymerase II.
• To determine if RNA polymerase II synthesis is an absolute requirement for in vivo 3' end processing and polyadenylation.

Main Methods:

• Engineering a viral RNA (vRNA) molecule with a replaced viral poly(A) site and a eukaryotic polyadenylation signal.
• Transcribing the engineered vRNA using the viral RNA polymerase.
• Analyzing the processing of the transcription product by the cellular 3' end processing machinery in vivo.

Main Results:

• The engineered vRNA molecule was successfully transcribed by the viral RNA polymerase.
• The cellular 3' end processing machinery recognized and processed the viral RNA transcript.
• This processing resulted in the addition of a poly(A) tail to the transcript, demonstrating in vivo polyadenylation.

Conclusions:

• Cellular polyadenylation machinery can process RNA transcripts synthesized by viral RNA polymerase, not just RNA polymerase II.
• RNA polymerase II synthesis is not an absolute requirement for efficient in vivo 3' end processing and polyadenylation.
• These findings challenge the current model of coupled transcription-polyadenylation for eukaryotic pre-mRNAs and broaden the understanding of RNA processing mechanisms.