Active RNA polymerases are localized within discrete transcription ‘factories’ in human nuclei

TLDR

The authors elongated nascent transcripts in permeabilized HeLa cells using Br‑UTP or biotin‑14‑CTP, then immunolabeled incorporation sites pre‑ or post‑embedding and visualized them by light or electron microscopy. They identified ~2,100 discrete nucleoskeleton‑attached sites surrounded by chromatin, each containing a cluster of ~20 engaged RNA polymerases and transcripts, supporting the existence of transcription factories where active polymerases are confined and nascent RNA is extruded.

Abstract

ABSTRACT Nascent transcripts in permeabilized HeLa cells were elongated by ∼30-2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in ∼2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription ‘factories’. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.