Keynote Conference - Interevent

Symp#26 RNA regulation - Canadian Society for Cell Biology
Chairs and speakers Jean Pierre Perrault and Carla Columbano
Impact of G-quadruplex structures on the human transcriptome
Jean-Pierre Perreault and Jean-Denis Beaudoin
Groupe ARN/RNA group, Département de biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke,
Sherbrooke, QC, J1H 5N4, Canada (Jean-Pierre.Perreault@USherbrooke.ca)
Given that greater than 90% of the human genome is expressed, it is logical to assume that post-transcriptional regulatory
mechanisms must be the primary means of controlling the flow of information from mRNA to protein. Guanine-rich nucleic acid
sequences can fold into non-canonical, four stranded helical structures called G-quadruplexes. Initially, we have developped a robust
approach that includes in silico, in vitro and in cellulo experiments permitting an in-depth evaluation of the global impact of Gquadruplexes
as translational repressors. Briefly, sequences including potential G-quadruplexes were selected within 9 distinct genes
encoding proteins involved in various biological processes. Six of these sequences were observed to fold into G-quadruplex structures
in vitro, all of which exhibited translational inhibition in cellulo when linked to a reporter gene. In addition, the impact of single
nucleotide polymorphism was shown to be important in the formation of G-quadruplexes located within the 5’-untranslated region of
an mRNA. Subsequently, the same approach was applied in order to study to evaluate the presence of G-quadruplex structures within
human 3'-UTRs. Specifically, two potential G-quadruplex sequences located in the 3'-UTR of the low density lipoprotein receptorrelated
protein 5 (LRP5) gene and the fragile X mental retardation autosomal homolog 1 (FXR1) gene were chaarcterized. Both of
these G-quadruplex structures increases by 2-fold the gene expression of a reporter gene by stimulating the polyadenylation of its
mRNA throughout an alternative site located downstream of the canonical site of their corresponding 3'-UTR. Sequence analysis, site
directed mutagenesis, miRNA regulation network analysis and G-quadruplex ligand experiments were performed to define rules
governing this phenomenon. In light of these results, we suggest that 3'-UTR G-quadruplexes can regulate alternative polyadenylation
sites, leading to the expression of shorter transcripts, and can interfer with the miRNA regulatory network of a specific mRNA. In light
of these results, the G-quadruplexes represent a class of RNA motif that is broadly distributed in the cellular transcriptome and have
important impact on mRNA species.
Identification of proteins regulating the RNA exosome
Carla Columbano Oliveira
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo
In eukaryotes, many posttranscriptional processing events are necessary for the synthesis of mature RNAs. mRNAs, tRNAs, rRNAs,
snRNAs and snoRNAs are processed through several steps that involve specific reactions between RNA and proteins. In fact, most
cellular RNAs are associated with proteins, forming ribonucleoprotein complexes (RNPs), which participate in different aspects of
gene expression.
The main focus of our laboratory has been the study of the posttranscriptional control of gene expression through the functional and
structural characterization of proteins that regulate processing of different types of RNA. We will show the identification and
functional characterization of Saccharomyces cerevisiae proteins that interact with and regulate the RNA exosome, a protein complex
involved in processing and degradation of all types of RNA. Nop53p and Nop8p are nucleolar proteins involved in the maturation of
the large ribosomal subunit and regulate the RNA exosome during this process in different ways. While Nop53p activates the
exosome, Nop8p inhibits it. These observations led to the hypothesis that the interactions between different proteins and the
exosome are responsible for directing the complex to its substrates, and for controlling its activity.
Inhibition of RNA Polymerase I as a Strategy to Treat Cancer
Megan J. Bywater 1 , Katherine M. Hannan 1 , Gretchen Poortinga 1 , Joanna C. Chan 1 , Elaine Sanij 1 , Nadine Hein 1 , Carleen Cullinane 1 ,
Denis Drygin 2 , William G. Rice 2 , Ricky W. Johnstone 1,3 , Grant A. McArthur 1,3 , Ross D. Hannan 1,3 and Richard B. Pearson 1,3 .
1 Division of Cancer Research, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Vic, Australia; 2 Cylene
Pharmaceuticals Inc., 5820 Nancy Ridge Drive, San Diego, CA 92121, USA; 3 Sir Peter MacCallum Department of Oncology, University
of Melbourne, Melbourne Australia.
Morphologic abnormalities of the nucleolus, the site of transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I), have
been recognized as diagnostic for cancer for more then a century. Furthermore, accelerated ribosome biogenesis is invariably
associated with malignant transformation. Nevertheless, a critical, unresolved question has been whether the accelerated ribosome
biogenesis responsible for the nucleolar changes is required for maintenance of the malignant phenotype.
Here we show that the PI3K/AKT pathway, deregulated in a high proportion of human tumours, is a critical regulator of ribosome
biogenesis. Constitutively active AKT is sufficient to drive rRNA synthesis, ribosome biogenesis and cell growth. Furthermore, AKT
cooperates with c-MYC to activate rRNA synthesis and ribosome biogenesis identifying the AKT/mTORC1/MYC network as a master
controller of cell growth. Consistent with this concept, AKT activity is required for maximal activation of rRNA synthesis and tumour
formation in the E�-Myc mouse model of Burkitt’s lymphoma (1). Our findings raise the exciting possibility that malignant diseases
characterized by unrestrained cellular growth may be vulnerable to therapeutic strategies that target ribosome biogenesis.
To directly test this hypothesis, we used genetic manipulation and a novel selective small molecule inhibitor of Pol I transcription (CX-
5461) (2), to provide the first definitive evidence that accelerated rDNA transcription and nucleolar integrity are necessary for
oncogenic activity in hematologic tumour cells. Further, we show that Pol I transcription can be targeted in vivo to therapeutically
treat tumors in both genetically engineered and xenograft models of lymphoma and leukemia through the non-genotoxic activation
of p53-dependent apoptosis, while sparing normal cells of hematological lineages. Thus, selective inhibition of Pol I transcription, a so
called ‘”house keeping” process, can serve as a novel therapeutic strategy for the treatment of cancer (3).
(1) Chan J.C, et al (2011) Science Signaling 4 (188), ra56, (2) Drygin, D et al., (2011) Cancer Res, 71(4):1418-3
(3) Bywater, M.J. et al (2012) Cancer Cell (accepted for publication)
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