Approach to Measuring the Effect of PARP1 on RNA Polymerase II Elongation Rates

Abstract

The rate of RNA polymerase II (RNAPII) transcriptional elongation plays a critical role in mRNA biogenesis, from transcription initiation to alternative splicing. As RNAPII moves along the DNA, it must read the DNA sequences wrapped up as chromatin. Thus, the structure of chromatin impedes the movement and speed at which RNAPII moves, presenting a crucial regulation to gene expression. Therefore, factors that bind and regulate the structure of chromatin will impact the rate of RNAPII elongation. We previously showed that PARP1 (poly-ADP-ribose polymerase 1) is one of such factors that bind and alter chromatin dynamics. We also showed that its alteration of chromatin structure modulates RNAPII processivity during transcriptional elongation. Here, we aim to understand how PARP1 alters RNAPII elongation kinetics genome wide.

Keywords: Chromatin structure; Nascent RNA-Seq; RNA polymerase II elongation; RNA transcription; Splicing.

Fig. 1

Generation of siRNA for PARP1 knockdown. (a) Lane 1, PCR ladder; Lane 2, amplified DNA of PARP1 gene from Drosophila genomic DNA; Lane 3, annealed and purified dsRNA. (b) Lane 1, PCR ladder; Lane 2, dsRNA as in Lane 3 of panel A; Lane 3, dsRNA digested to yield small siRNAs for PARP1 knockdown. Short run of samples on a 4% Nusieve agarose gel to capture the shorter siRNA samples. A longer run results in diffused bands

Fig. 2

Validation of PARP1 knockdown. PARP1 was knocked down with siRNA as above. (a) Protein extracts were quantified, run on SDS PAGE and western blotted for PARP1 antibodies. (b) RNA was purified from cells and converted to cDNA, and the level of PARP1 transcript expression analyzed by qRT-PCR. *Mean of n = 3 ± SEM, p < 0.05 by T-test

Fig. 3

Overall workflow of protocol. (a) S2 Drosophila cells were grown in T75 flasks and treated with DRB for 4 h to stop transcription. (b) Cells were washed with 1X PBS to remove the DRB. (c) To label newly synthesized (nascent) RNA, cells now in growth media were treated with 4sU before being collected. Time point 0 min = 4 h of DRB and collected immediately after DRB removal. Time point 4 min = 4 h of DRB and collection 4 min after DRB removal. Time point 8 min = 4 h of DRB and collection 8 min after DRB removal. Time point 16 min = 4 h of DRB and collection after 16 min. (d) Samples were quenched with Trizol and total RNA purified. (e) 4sU-labeled RNA was biotinylated, purified with streptavidin beads, and subjected to deep sequencing