The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

Abstract

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)-dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40-60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II-dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid-protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex

“…However, Western blotting analysis using the anti-FLAG antibody could not detect the FLAG-tagged RPA40 in a fraction immunoprecipitated with an anti-WRN antibody, suggesting that large amount of WRN localized in the nucleolus is free from the RPI complex or the binding a nity between WRN and the RPI complex is relatively low. Balajee et al (1999) reported that RPII-speci®c transcription in WS B-lymphoblastoid cells transformed with SV40 is 40 ± 60% lower than that of cells from healthy individuals, and that RPII-dependent transcription is stimulated by adding puri®ed wild type, but not mutant WRN, in an in vitro assay using permeabilized cells. Recently, Kadener et al (2001) reported that SV40 large T-antigen activates template replication only twofold but transcription 25-fold.…”

“…In addition, we found no marked di erence in RPI-and RPII-dependent transcription activities between permeabilized normal and WS ®broblasts (data not shown). Moreover it is possible that treatment required for permeabilization of cells might have caused subnuclear relocation and loss of WRN in the experiments by Balajee et al (1999). To resolve the issue, we monitored the transcription e ciency of a primary culture of normal and WS ®broblasts by measuring [ 3 H]-uridine incorporation into a total RNA fraction avoiding the process of membrane permeabilization.…”

“…These results suggest that the major component contributing the increase in transcription of total RNA in response to expression of exogenous wtWRN in WS cells must be rRNA rather than mRNA. Although Balajee et al (1999) reported complementation of the reduced RPIIdependent transcription in WS cells with nuclear extracts from HeLa cells, they used as a template for transcription, chromatin prepared from permeabilized WS cells that were depleted of soluble proteins. Thus, unlike the data described here, their complementation experiments were not carried out in intact cells.…”

Gene expression profiling in Werner syndrome closely resembles that of normal aging

“…The mechanisms by which the biochemical deficiencies resulting from WRN mutations lead to the characteristic pathology of the syndrome are not yet understood. It has been hypothesized that several WS phenotypes are secondary consequences of aberrant gene expression (6) and that a transcription defect may be crucial to the development of the syndrome (7). Increasing evidence suggests that WRN has a role in transcription.…”

High expression stability of microtubule affinity regulating kinase 3 (MARK3) makes it a reliable reference gene

“…These suggest that genes with their resulting protein products that are involved in translation of mRNA are more likely to be constitutively expressed at a constant level as varying availability of proteins in the translational process may result in variability in translational efficiency (22). Increase in translational inefficiency has been implicated in the molecular aging process (23), suggesting that variability in translational efficiency is not desirable and should be minimised.…”