Xavier Darzacq

In vivo dynamics of RNA polymerase II transcription

We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min−1, much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo.

Cajal body-specific small nuclear RNAs: a novel class of 2′-O-methylation and pseudouridylation guide RNAs

Cajal (coiled) bodies are conserved subnuclear organelles that are present in the nucleoplasm of both animal and plant cells. Although Cajal bodies were first described nearly 100 years ago, their function has remained largely speculative. Here, we describe a novel class of human small nuclear RNAs that localize specifically to Cajal bodies. The small Cajal body‐ specific RNAs (scaRNAs) are predicted or have already been demonstrated to function as guide RNAs in site‐specific synthesis of 2′‐O‐ribose‐methylated nucleotides and pseudouridines in the RNA polymerase II‐transcribed U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs). Our results provide strong support for the idea that the Cajal body, this mysterious nuclear organelle, provides the cellular locale for post‐transcriptional modification of spliceosomal snRNAs.

Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm

Biogenesis of functional spliceosomal small nuclear RNAs (snRNAs) includes the post‐transcriptional covalent modification of numerous internal nucleotides. We have recently demonstrated that synthesis of 2′‐O‐methylated nucleotides and pseudouridines in the RNA polymerase II‐synthesized Sm snRNAs is directed by sequence‐specific guide RNAs. Here, we provide evidence supporting the notion that modification of Sm snRNAs occurs in nucleoplasmic Cajal bodies (CBs), where modification guide RNAs accumulate. We show that short fragments of Sm snRNAs are correctly and efficiently modified when targeted to CBs, but not when these same fragments are targeted to the nucleolus. We also demonstrate that internal modification of the U2 snRNA occurs exclusively after nuclear import of the newly assembled Sm snRNP from the cytoplasm. Finally, we show that p80 coilin, the CB marker protein, is not required for snRNA modification. In coilin knockout cells, Sm snRNAs and their modification guide RNAs colocalize in residual CBs, which do not stockpile fibrillarin and fail to recruit the U3 small nucleolar RNA.

A transgenic mouse for in vivo detection of endogenous labeled mRNA.

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3′ untranslated region of the essential ββ-actin gene. As β-actin–MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.

Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells

Pol II Micro Clusters In higher eukaryotes, messenger RNA (mRNA) synthesis is thought to involve foci of clustered RNA polymerase II (Pol II) called transcription factories. However, clustered Pol II have not been resolved in living cells, raising the debate about their existence in vivo and what role, if any, they play in nuclear organization and regulation of gene expression. Cisse et al. (p. 664, published online 4 July; see the Perspective by Rickman and Bickmore) developed single-molecule in vivo analyses revealing the distribution and dynamics of Pol II clustering in living cells. Pol II clusters were smaller than the diffraction limit (<250 nm). Transient dynamics of the Pol II clusters, and correlation with changes in transcription, pointed to a role in transcription initiation rather than in elongation. A single-cell quantitative method reveals changes in the distribution of proteins with single-molecule sensitivity. [Also see Perspective by Rickman and Bickmore] Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell’s ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

A common sequence motif determines the Cajal body‐specific localization of box H/ACA scaRNAs

Post‐transcriptional synthesis of 2′‐O‐methylated nucleotides and pseudouridines in Sm spliceosomal small nuclear RNAs takes place in the nucleoplasmic Cajal bodies and it is directed by guide RNAs (scaRNAs) that are structurally and functionally indistinguishable from small nucleolar RNAs (snoRNAs) directing rRNA modification in the nucleolus. The scaRNAs are synthesized in the nucleoplasm and specifically targeted to Cajal bodies. Here, mutational analysis of the human U85 box C/D‐H/ACA scaRNA, followed by in situ localization, demonstrates that box H/ACA scaRNAs share a common Cajal body‐specific localization signal, the CAB box. Two copies of the evolutionarily conserved CAB consensus (UGAG) are located in the terminal loops of the 5′ and 3′ hairpins of the box H/ACA domains of mammalian, Drosophila and plant scaRNAs. Upon alteration of the CAB boxes, mutant scaRNAs accumulate in the nucleolus. In turn, authentic snoRNAs can be targeted into Cajal bodies by addition of exogenous CAB box motifs. Our results indicate that scaRNAs represent an ancient group of small nuclear RNAs which are localized to Cajal bodies by an evolutionarily conserved mechanism.

Nucleolar Factors Direct the 2′-O-Ribose Methylation and Pseudouridylation of U6 Spliceosomal RNA

ABSTRACT The nucleolus has long been known as a functionally highly specialized subnuclear compartment where synthesis, posttranscriptional modification, and processing of cytoplasmic rRNAs take place. In this study, we demonstrate that the nucleolus contains all thetrans-acting factors that are responsible for the accurate and efficient synthesis of the eight 2′-O-methylated nucleotides and three pseudouridine residues carried by the mammalian U6 spliceosomal small nuclear RNA. Factors mediating the formation of pseudouridine residues in the U3 small nucleolar RNA are also present and functionally active in the nucleolus. For selection of the correct target nucleotides in the U6 and U3 RNAs, the nucleolar 2′-O-methylation and pseudouridylation factors rely on short sequences located around the target nucleotide to be modified. This observation further underscores a recently proposed role for small nucleolar guide RNAs in the 2′-O-methylation of the U6 spliceosomal RNA (K. T. Tycowski, Z.-H. You, P. J. Graham, and J. A. Steitz, Mol. Cell 2:629–638, 1998). We demonstrate that a novel 2′-O-methylated nucleotide can be generated in the yeast U6 RNA by use of an artificial 2′-O-methylation small nucleolar guide RNA. We also show that a short fragment of the 5.8S rRNA, when expressed as part of the human U6 RNA, is faithfully 2′-O-methylated and pseudouridylated. These results are most consistent with a trafficking pathway in which the U6 spliceosomal RNA cycles through the nucleolus to undergo nucleolar RNA-directed modifications.

The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing

Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase.

Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells

Mammalian H/ACA RNPs are essential for ribosome biogenesis, premessenger RNA splicing, and telomere maintenance. These RNPs consist of four core proteins and one RNA, but it is not known how they assemble. By interrogating the site of H/ACA RNA transcription, we dissected their biogenesis in single cells and delineated the role of the non-core protein NAF1 in the process. NAF1 and all of the core proteins except GAR1 are recruited to the site of transcription. NAF1 binds one of the core proteins, NAP57, and shuttles between nucleus and cytoplasm. Both proteins are essential for stable H/ACA RNA accumulation. NAF1 and GAR1 bind NAP57 competitively, suggesting a sequential interaction. Our analyses indicate that NAF1 binds NAP57 and escorts it to the nascent H/ACA RNA and that GAR1 then replaces NAF1 to yield mature H/ACA RNPs in Cajal bodies and nucleoli.