An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes

A Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei. Prodigious amounts of VSG mRNA (~7-10% total) are generated from a single RNA polymerase I (Pol I) transcribed VSG expression site (ES), necessitating extremely high levels of localised splicing. We show that splicing is required for processive ES transcription, and describe novel ES-associated T. brucei nuclear bodies. In bloodstream form trypanosomes, the expression site body (ESB), spliced leader array body (SLAB), NUFIP body and Cajal bodies all frequently associate with the active ES. This assembly of nuclear bodies appears to facilitate the extraordinarily high levels of transcription and splicing at the active ES. In procyclic form trypanosomes, the NUFIP body and SLAB do not appear to interact with the Pol I transcribed procyclin locus. The congregation of a restricted number of nuclear bodies at a single active ES, provides an attractive mechanism for how monoallelic ES transcription is mediated.

Cryo-EM structures of human RNA polymerase I

RNA polymerase I (Pol I) specifically synthesizes ribosomal RNA. Pol I upregulation is linked to cancer, while mutations in the Pol I machinery lead to developmental disorders. Here we report the cryo-EM structure of elongating human Pol I at 2.7 Å resolution. In the exit tunnel, we observe a double-stranded RNA helix that may support Pol I processivity. Our structure confirms that human Pol I consists of 13 subunits with only one subunit forming the Pol I stalk. Additionally, the structure of human Pol I in complex with the initiation factor RRN3 at 3.1 Å resolution reveals stalk flipping upon RRN3 binding. We also observe an inactivated state of human Pol I bound to an open DNA scaffold at 3.3 Å resolution. Lastly, the high-resolution structure of human Pol I allows mapping of disease-related mutations that can aid understanding of disease etiology.

Defining the Influence of the A12.2 Subunit on Transcription Elongation and Termination by RNA Polymerase I In Vivo

Saccharomyces cerevisiae has approximately 200 copies of the 35S rDNA gene, arranged tandemly on chromosome XII. This gene is transcribed by RNA polymerase I (Pol I) and the 35S rRNA transcript is processed to produce three of the four rRNAs required for ribosome biogenesis. An intergenic spacer (IGS) separates each copy of the 35S gene and contains the 5S rDNA gene, the origin of DNA replication, and the promoter for the adjacent 35S gene. Pol I is a 14-subunit enzyme responsible for the majority of rRNA synthesis, thereby sustaining normal cellular function and growth. The A12.2 subunit of Pol I plays a crucial role in cleavage, termination, and nucleotide addition during transcription. Deletion of this subunit causes alteration of nucleotide addition kinetics and read-through of transcription termination sites. To interrogate both of these phenomena, we performed native elongating transcript sequencing (NET-seq) with an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy across the 35S gene and the IGS. Compared to wild-type (WT), we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Relative occupancy of rpa12Δ Pol I increased upstream of the promoter-proximal Reb1 binding site and dropped significantly downstream, implicating this site as a third terminator for Pol I transcription. Collectively, these high-resolution results indicate that the A12.2 subunit of Pol I plays an important role in transcription elongation and termination.

Nucleolar translocation of human DNA topoisomerase II by ATP depletion and its disruption by the RNA polymerase I inhibitor BMH-21

DNA topoisomerase II (TOP2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two TOP2 proteins, TOP2A and TOP2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of TOP2B, but the molecular details of subnuclear distributions, particularly of TOP2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human TOP2A and TOP2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the TOP2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the TOP2 poison etoposide was less effective, indicating the therapeutic relevance of TOP2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of TOP2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

Structure of the human RNA polymerase I elongation complex

Eukaryotic RNA polymerase I (Pol I) transcribes ribosomal DNA and generates RNA for ribosome synthesis. Pol I accounts for the majority of cellular transcription activity and dysregulation of Pol I transcription leads to cancers and ribosomopathies. Despite extensive structural studies of yeast Pol I, structure of human Pol I remains unsolved. Here we determined the structures of the human Pol I in the pre-translocation, post-translocation, and backtracked states at near-atomic resolution. The single-subunit peripheral stalk lacks contacts with the DNA-binding clamp and is more flexible than the two-subunit stalk in yeast Pol I. Compared to yeast Pol I, human Pol I possesses a more closed clamp, which makes more contacts with DNA. The Pol I structure in the post-cleavage backtracked state shows that the C-terminal zinc ribbon of RPA12 inserts into an open funnel and facilitates “dinucleotide cleavage” on mismatched DNA–RNA hybrid. Critical disease-associated mutations are mapped on Pol I regions that are involved in catalysis and complex organization. In summary, the structures provide new sights into human Pol I complex organization and efficient proofreading.

Nucleolar translocation of human DNA topoisomerase II by ATP depletion and its disruption by the RNA polymerase I inhibitor BMH-21

DNA topoisomerase II (Top2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two Top2 proteins, Top2A and Top2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of Top2B, but the molecular details of subnuclear distributions, particularly of Top2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human Top2A and Top2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the Top2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the Top2 poison etoposide was less effective, indicating the therapeutic relevance of Top2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of Top2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

lncRNA SLERT controls phase separation of FC/DFCs to facilitate Pol I transcription

RNA polymerase I (Pol I) transcription takes place at the border of the fibrillar center (FC) and the dense fibrillar component (DFC) in the nucleolus. Here, we report that individual spherical FC/DFC units are coated by the DEAD-box RNA helicase DDX21 in human cells. The long noncoding RNA (lncRNA) SLERT binds to DDX21 RecA domains to promote DDX21 to adopt a closed conformation at a substoichiometric ratio through a molecular chaperone–like mechanism resulting in the formation of hypomultimerized and loose DDX21 clusters that coat DFCs, which is required for proper FC/DFC liquidity and Pol I processivity. Our results suggest that SLERT is an RNA regulator that controls the biophysical properties of FC/DFCs and thus ribosomal RNA production.