Imaging of native transcription and transcriptional dynamics in vivo using a tagged Argonaute protein

A flexible method to image unmodified transcripts and transcription in vivo would be a valuable tool to understand the regulation and dynamics of transcription. Here, we present a novel approach to follow native transcription, with fluorescence microscopy, in live C. elegans. By using the fluorescently tagged Argonaute protein NRDE-3, programmed by exposure to defined dsRNA to bind to nascent transcripts of the gene of interest, we demonstrate transcript labelling of multiple genes, at the transcription site and in the cytoplasm. This flexible approach does not require genetic manipulation, and can be easily scaled up by relying on whole-genome dsRNA libraries. We apply this method to image the transcriptional dynamics of two transcription factors: ttx-3 (a LHX2/9 orthologue) in embryos, and hlh-1 (a MyoD orthologue) in larvae, respectively involved in neuronal and muscle development.

The spatial distributions of pre-mRNAs suggest post-transcriptional splicing of specific introns within endogenous genes

Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. Measuring the timing of splicing relative to the transcription of nascent RNA has yielded conflicting interpretations. Biochemical fractionation suggests that RNA is spliced primarily during the process of transcription, but imaging of nascent RNA suggests that splicing happens after the process of transcription has been completed. We use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the delay between when an intron is transcribed and when it is spliced out of a pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit some post-transcriptional splicing, and that introns can be spliced in any order. We also show that completely synthesized RNA move slowly through a transcription site proximal zone while they undergo additional splicing and potentially other processing after transcription is completed. In addition, upon leaving this zone, some genes’ transcripts localize to speckles during the process of splicing but some appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Taken together, our observations suggest that the regulation of the timing and localization of splicing is specific to individual introns, as opposed to the previously surmised immediate excision of introns after transcription.

TGF-β1 induces Fstl1 via the Smad3-c-Jun pathway in lung fibroblasts

Transforming growth factor (TGF)-β1 has long been regarded as a central mediator of tissue fibrosis. Follistatin-like 1 (Fstl1) is a crucial profibrotic glycoprotein that is upregulated in fibrotic lung tissues, and it promotes fibrogenesis via facilitating TGF-β signaling. Here we examined the signaling pathway by which TGF-β1 upregulates Fstl1 expression in mouse pulmonary fibroblasts. TGF-β1 regulated Fstl1 expression at both the transcriptional and translational levels. Although TGF-β1 rapidly activated the Smad, MAPK, and Akt pathways in lung fibroblasts, only Smad2/3 inhibition eliminated TGF-β1−induced Fstl1 expression. Analysis of the luciferase reporter activity identified a functional c-Jun transcription site in the Fstl1 promoter. Our results suggested a critical role for the Smad3-c-Jun pathway in the regulation of Fstl1 expression by TGF-β1 during fibrogenesis.

Transcription regulation during stable elongation by a reversible halt of RNA polymerase II

Regulation of RNA polymerase II (RNAPII) during transcription is essential for controlling gene expression. Here we report that the transcriptional activity of RNAPII at the Balbiani ring 2.1 gene could be halted during stable elongation in salivary gland cells of Chironomus tentans larvae for extended time periods in a regulated manner. The transcription halt was triggered by heat shock and affected all RNAPII independently of their position in the gene. During the halt, incomplete transcripts and RNAPII remained at the transcription site, the phosphorylation state of RNAPII was unaltered, and the transcription bubbles remained open. The transcription of halted transcripts was resumed upon relief of the heat shock. The observed mechanism allows cells to interrupt transcription for extended time periods and rapidly reactivate it without the need to reinitiate transcription of the complete gene. Our results suggest a so-far-unknown level of transcriptional control in eukaryotic cells.

Sp100A promotes chromatin decondensation at a cytomegalovirus-promoter–regulated transcription site

Promyelocytic leukemia nuclear bodies (PML-NBs)/nuclear domain 10s (ND10s) are nuclear structures that contain many transcriptional and chromatin regulatory factors. One of these, Sp100, is expressed from a single-copy gene and spliced into four isoforms (A, B, C, and HMG), which differentially regulate transcription. Here we evaluate Sp100 function in single cells using an inducible cytomegalovirus-promoter–regulated transgene, visualized as a chromatinized transcription site. Sp100A is the isoform most strongly recruited to the transgene array, and it significantly increases chromatin decondensation. However, Sp100A cannot overcome Daxx- and α-thalassemia mental retardation, X-linked (ATRX)–mediated transcriptional repression, which indicates that PML-NB/ND10 factors function within a regulatory hierarchy. Sp100A increases and Sp100B, which contains a SAND domain, decreases acetyl-lysine regulatory factor levels at activated sites, suggesting that Sp100 isoforms differentially regulate transcription by modulating lysine acetylation. In contrast to Daxx, ATRX, and PML, Sp100 is recruited to activated arrays in cells expressing the herpes simplex virus type 1 E3 ubiquitin ligase, ICP0, which degrades all Sp100 isoforms except unsumoylated Sp100A. The recruitment Sp100A(K297R), which cannot be sumoylated, further suggests that sumoylation plays an important role in regulating Sp100 isoform levels at transcription sites. This study provides insight into the ways in which viruses may modulate Sp100 to promote their replication cycles.

Splicing- and cleavage-independent requirement of RNA polymerase II CTD for mRNA release from the transcription site

Eukaryotic cells have a surveillance mechanism that identifies aberrantly processed pre-mRNAs and prevents their flow to the cytoplasm by tethering them near the site of transcription. Here we provide evidence that mRNA release from the transcription site requires the heptad repeat structure of the C-terminal domain (CTD) of RNA polymerase II. The mammalian CTD, which is essential for normal co-transcriptional maturation of mRNA precursors, comprises 52 heptad repeats. We show that a truncated CTD containing 31 repeats (heptads 1–23, 36–38, and 48–52) is sufficient to support transcription, splicing, cleavage, and polyadenylation. Yet, the resulting mRNAs are mostly retained in the vicinity of the gene after transcriptional shutoff. The retained mRNAs maintain the ability to recruit components of the exon junction complex and the nuclear exosome subunit Rrp6p, suggesting that binding of these proteins is not sufficient for RNA release. We propose that the missing heptads in the truncated CTD mutant are required for binding of proteins implicated in a final co-transcriptional maturation of spliced and 3′ end cleaved and polyadenylated mRNAs into export-competent ribonucleoprotein particles.