Abstract

We have focused on the mechanism of long noncoding (lnc)RNA synthesis in mammalian cell lines. Many of these lncRNA are associated with protein coding (pc-) genes and include eRNA that derive from pc-gene enhancer regions, pc-gene promoter associated antisense transcripts, pc-gene antisense transcripts and separate long intergenic noncoding (linc)RNA. Remarkably even though lncRNA are transcribed by the same RNA polymerase II (Pol II) transcriptional apparatus, both the beginning and end of lncRNA transcription units (TUs) appear to use radically different mechanisms. 1) We show that lncRNA promoters are often associated with R-loop structures generally formed by more abundant pc-gene transcripts. The displaced single strand DNA acts as a de novo Pol II promoter inducing antisense transcription from the single stranded DNA region (Tan-Wong et al. Mol. Cell. 2019). 2) lncRNA in general are only weakly 3’ end processed by cleavage and polyadenylation activity (normally associated with pc-gene transcripts). Instead the Integrator complex appears instrumental in promoting length restricted polyA minus lncRNA (Nojima et al. Mol. Cell. 2018). The consequence of these non-pc-gene mechanisms to define either end of lncRNA TUs is the likely cause of their rapid degradation before release from the chromatin template (Schlackow et al. Mol. Cell. 2017). Functional lncRNA must evade this nuclear surveillance mechanism to allow sufficient accumulation to effect particular biological processes. We are developing a new approach to sequence full length nascent transcripts with their 5’ ends derived from transcription start sites and 3’ ends within the elongating RNA polymerase complex. This method is an extension of our original mNET-seq approach (Nojima et al. Cell. 2015) and vividly reveals by single molecule Nanopore sequencing the coupled processing of nascent transcripts.

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