Bruno Lemieux

The 3D nuclear organization of telomeres marks the transition from Hodgkin to Reed-Sternberg cells

To get an insight into the transition from mononuclear Hodgkin cells (H cells) to diagnostic multinuclear Reed–Sternberg cells (RS cells), we performed an analysis of the three-dimensional (3D) structure of the telomeres in the nuclei of the Hodgkin cell lines HDLM-2, L-428, L-1236 and lymph node biopsies of patients with Hodgkins disease. Cellular localization of key proteins of the telomere-localized shelterin complex, the mitotic spindle and double-stranded DNA breaks was also analyzed. RS cells show significantly shorter and significantly fewer telomeres in relation to the total nuclear volume when compared with H cells; in particular, telomere-poor ‘ghost’ nuclei are often adjacent to one or two nuclei displaying huge telomeric aggregates. Shelterin proteins are mainly cytoplasmic in both H and RS cells, whereas double-stranded DNA breaks accumulate in the nuclei of RS cells. In RS cells, multipolar spindles prevent proper chromosome segregation. In conclusion, a process of nuclear disorganization seems to initiate in H cells and further progresses when the cells turn into RS cells and become end-stage tumor cells, unable to divide further because of telomere loss, shortening and aggregate formation, extensive DNA damage and aberrant mitotic spindles that may no longer sustain chromosome segregation. Our findings allow a mechanistic 3D understanding of the transition of H to RS cells.

TCERG1 Regulates Alternative Splicing of the Bcl-x Gene by Modulating the Rate of RNA Polymerase II Transcription

ABSTRACT Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the proapoptotic Bcl-xS 5′ splice site.

A Function for the hnRNP A1/A2 Proteins in Transcription Elongation

The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes.

The yeast telomerase module for telomere recruitment requires a specific RNA architecture

Telomerases are ribonucleoprotein (RNP) reverse transcriptases. While telomerases maintain genome stability, their composition varies significantly between species. Yeast telomerase RNPs contain an RNA that is comparatively large, and its overall folding shows long helical segments with distal functional parts. Here we investigated the essential stem IVc module of the budding yeast telomerase RNA, called Tlc1. The distal part of stem IVc includes a conserved sequence element CS2a and structurally conserved features for binding Pop1/Pop6/Pop7 proteins, which together function analogously to the P3 domains of the RNase P/MRP RNPs. A more proximal bulged stem with the CS2 element is thought to associate with Est1, a telomerase protein required for telomerase recruitment to telomeres. Previous work found that changes in CS2a cause a loss of all stem IVc proteins, not just the Pop proteins. Here we show that the association of Est1 with stem IVc indeed requires both the proximal bulged stem and the P3 domain with the associated Pop proteins. Separating the P3 domain from the Est1 binding site by inserting only 2 base pairs into the helical stem between the two sites causes a complete loss of Est1 from the RNP and hence a telomerase-negative phenotype in vivo. Still, the distal P3 domain with the associated Pop proteins remains intact. Moreover, the P3 domain ensures Est2 stability on the RNP independently of Est1 association. Therefore, the Tlc1 stem IVc recruitment module of the RNA requires a very tight architectural organization for telomerase function in vivo.