Luiz Otavio F. Penalva, Ph.D., Assistant Professor
Universidad Autonoma de Madrid, Spain
1996

CCRI (210) 562-9049
PENALVA@UTHSCSA.EDU

Dr. Luiz Otavio Penalva joined the Department of Cellular and Structural Biology and the Children's Cancer Research Institute in July 2004.

Post-transcriptional regulation of gene expression plays a pivotal role in determining the quality and quantity of protein expression. RNA binding proteins (RBPs) are the principal regulators of post-transcriptional events, which include pre-mRNA splicing, mRNA transport and localization, mRNA stability and translation. Our lab applies genomic technologies to characterize RNA sub-populations associated with specific RNA binding proteins in an effort to provide a better understanding of biological processes like tumorigenesis.

Dissecting gene expression of tumors and complex tissues with RNA binding proteins
The gene expression profile of a whole tumor corresponds to the sum of individual profiles of different cell types (i.e.: endothelial cells, T-cells, fibroblasts, cancer cells, etc) that are interdependent and exchange biochemical signals as a means of cell-cell communication. In order to better understand heterotypic cell-cell communication and its role in tumorigenesis, one has to be able to: 1) characterize independent gene expression profiles for each cell type within the tumor or tissue and 2) to determine how this cell-cell communication affects the mechanisms that regulate gene expression in each individual cell type.

Our lab is working on the development of a methodology that will allow the recovery from a complex tissue or tumor of cell type specific RNA sub populations associated to tagged RNA-binding proteins.

Figure 1 - Gene expression profiles of tumors and complex tissues. (click on Figure 1 to view larger image)

A) Transcriptomic analysis can be performed by using total RNA extracted from whole tumors. Technologies like microdissection can be utilized to isolate cell populations from complex tissues, allowing RNA preparation followed by transcriptomic analysis.

B) Tagged or endogenous RNA-protein complexes (RNPs) can be isolated directly from a particular cell type present in a complex tissue such as a tumor by (immuno)precipitation. The RNAs associated with the targeted mRNP complexes are then characterized using an en masse assay such as a cDNA microarray.

Dissecting the function of RNA binding protein/RNA binding associated proteins with genomic analysis
The Drosophila sex determination pathway provides one of the best model systems for post-transcriptional regulation studies. Several members of this pathway are RNA binding proteins or RNA binding associated proteins whose function is to regulate the splicing or/and translation of their downstream target genes. Despite the apparent differences between the Drosophila and the mammalian sex determination systems, some strategies seemed to be conserved along the evolution. For instance, several genes involved in mammalian sex determination, initially described as transcription factors, have been found to posses also RNA binding capacity and the potential to function as post-transcriptional regulators.

One of these genes encodes for the Wilms' tumor 1 protein (WT1). WT1 is necessary for genitor-urinary development and, its mutation is associated with the Wilms' tumor, a common type of pediatric kidney cancer. One isoform of WT1 has been shown to have RNA binding properties, to be associated and localized with splicing factors.

Curiously, a factor that interacts with WT1, Wilms' tumor associated protein (WTAP), turns to be the mammalian homologue of the Drosophila sex determination gene female-lethal-(2)-d [fl(2)d]. This gene encodes a nuclear protein required for proper splicing regulation of the Sex-lethal (Sxl) and transformer (tra) RNAs. The lack of identity with other known splicing factors puts Fl(2)d/WTAP in a novel class of splicing co-regulators.

Figure 2 (click on Figure 2 to view larger image) - Sex determination cascades in D. melanogaster and mammals - D. melanogaster In females: Sxl regulates the splicing of its own pre-mRNA, a positive feedback loop. The products of the genes fl(2)d, vir, snf and spf45 are also necessary for this splicing regulation. Sxl downstream target is the gene tra; splicing control by Sxl allows the production of a functional protein product. Tra forms a heterodimer with the Ttransformer-2 (Tra-2) protein that modulates the splicing of two genes: double sex (dsx) and fruitless (fru). The generated sex specific products control the expression of target genes necessary for female sex differentiation and behavior. In males: no Sxl protein is produced. As a consequence, tra RNA follows a different splicing pattern and no function product is generated. fru and dsx produce male specific transcripts. FruM and DsxM control the expression of target genes necessary for male sex differentiation and behavior. The gene dissatisfaction (dsf ) is implicated in both male and female sexual behavior. CNS -Central nervous system. Mammals WT1 upregulates SRY. WTAP is required for WT1 function. SRY represses the function of unknown gene Z (candidates are in parenthesis). Inhibition of these genes allows expression of testis differentiation genes.

Very little is known about the mechanism(s) by which Fl(2)d/WTAP and WT1 control splicing. To gain more insight into the function of those proteins as post-transcriptional regulators, we are working on: 1) gene expression analysis of Fl(2)d/WTAP and WT1 using genome based technologies in different cell lines and tissues at different developmental stages in Drosophila and mouse; 2) the development of in vivo and in vitro systems to explore the mechanism(s) by which Fl(2)d/WTAP and WT1 control the splicing of their target genes.

Publications:
L. O. F. Penalva, M. Fernanda Ruiz, Angeles Ortega, B. Granadino, L. Vicente, C. Segarra, J. Valc.rcel and L. S.nchez (2000) - The Drosophila fl(2)d gene, required for female-specific splicing of Sxl and tra pre-mRNAs, encodes a novel nuclear protein with a HQ-rich domain. Genetics 155(1): 129-139.

L.O F. Penalva, M.J. Lallena and J.Valc.rcel (2001) Switch in 3' splice site recognition between exon definition and splicing catalysis is important for Sex-lethal autoregulation. Mol. Cell Biol. 21(6): 1986-1996.

L.O. F. Penalva and L. S.nchez (2003) The RNA binding protein Sex-lethal (Sxl) and the control of Drosophila sex determination and dosage compensation. Micro. Mol. Biol. Rev. 67(3): 343-359.

Penalva LO, Burdick MD, Lin SM, Sutterluety H, Keene JD. RNA-binding proteins to assess gene expression states of co-cultivated cells in response to tumor cells. Mol Cancer. 2004 Sep 07;3(1):24.

Penalva LO, Keene JD. Biotinylated tags for recovery and characterization of ribonucleoprotein complexes. Biotechniques. 2004 Oct;37(4):604, 606, 608-10.