How Cell Stress and 3′ End Alterations Control the Metabolism of a Cellular Non‐Coding RNA

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RNA Polymerase II (Pol II) canonically acts as a DNA‐dependent RNA polymerase (DdRP), using double stranded DNA to synthesize protein‐encoding mRNAs and some non‐coding RNAs. Pol II also has RNA‐dependent RNA polymerase activity (RdRP), which uses an RNA as a template to synthesize RNA. An example of Pol II RdRP activity is the 3′ end extension of the non‐coding B2 RNA to generate extended B2 (eB2) RNA. B2 RNA binds to Pol II and is a substrate for a Pol II‐dependent 18 nucleotide RdRP‐catalyzed extension to form eB2 RNA. eB2 RNA can be detected in cells and has a reduced half‐life as compared to B2 RNA.B2 RNA is transcribed by Pol III from Short Interspersed Elements (SINEs), which exist in over 350,000 copies in the mouse genome due to retrotransposition. Upon cellular stress, transcription of non‐coding B2 RNAs from B2 SINEs is greatly increased, thereby increasing the likelihood of retrotransposition of B2 SINEs. The RdRP activity of Pol II could control the levels of B2 RNA post‐transcriptionally by generating eB2 RNA to promote its degradation, thereby impacting the frequency of retrotransposition.To study the effects of 3′ end extension and cellular stress on the lifecycle and metabolism of B2 RNA we are developing a heterologous expression system. Because the B2 SINE sequence is embedded in many mouse mRNA transcripts (e.g. UTRs and introns), it is impossible to perform hybridization and PCR‐based experiments to distinguish between a Pol III‐derived B2 RNA and those sequences within mRNA transcripts. To overcome this, we express B2 and eB2 RNA in human cells, which do not contain B2 SINEs, using a human Pol III promoter. The expressed RNAs are the correct size, have the correct 5′ ends, have the correct 3′ secondary structure, and exogenous eB2 has decreased stability compared to B2 RNA. Therefore, the heterologous system mimics characteristics of B2 RNA and eB2 RNA in mouse cells.Using this system, ongoing experiments are investigating how extension alters the cellular localization of B2 RNA and retrotransposition efficiency in unstressed and stressed cells. Altogether, these experiments will determine the interplay between B2 RNA expression, Pol II RdRP extension, cellular localization, and cellular stress on the life cycle of this ncRNA.Support or Funding InformationR01 GM68414 T32GM008759This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.