Molecular biology and medicine at the end of the XXth century

This paper reviews basic concepts of modern molecular biology with the premise that its influence in today's medicine is so important that its knowledge cannot remain limited to a few experts. I first analyze the overall structure and organization of human genes, their split nature and the flow of genetic information from DNA to protein. The role of transcriptional control in the regulation of gene expression and cell differentiation is described by introducing experimental examples that define the importance of "master" genes. Basic concepts of genetic engineering, the generation of transgenic and knock out animals and the uses of molecular biology in clinical diagnosis, paternity tests and forensic medicine are presented. Finally, I discuss the possibilities of gene therapy and the fantasies and realities of transgenesis and cloning by nuclear transplant in humans.Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Playing inside the genes: Intragenic histone acetylation after membrane depolarization of neural cells opens a path for alternative splicing regulation

Regulation of alternative splicing is coupled to transcription quality, the polymerase elongation rate being an important factor in modulating splicing choices. In a recently published work, we provide evidence that intragenic histone acetylation patterns can be affected by neural cell excitation in order to regulate alternative splicing of the neural cell adhesion molecule (NCAM) mRNA. This example illustrates how an extracellular stimulus can influence transcription-coupled alternative splicing, strengthening the link between chromatin structure, transcriptional elongation and mRNA processing. ©2009 Landes Bioscience.Fil:Schor, I.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Identification of the cellular targets of the transcription factor TCERG1 reveals a prevalent role in mrna processing

The transcription factor TCERG1 (also known as CA150) associates with RNA polymerase II holoenzyme and alters the elongation efficiency of reporter transcripts. TCERG1 is also found as a component of highly purified spliceosomes and has been implicated in splicing. To elucidate the function of TCERG1, we used short interfering RNA-mediated knockdown followed by en masse gene expression analysis to identify its cellular targets. Analysis of data from HEK293 and HeLa cells identified high confidence targets of TCERG1. We found that targets of TCERG1 were enriched in microRNA-binding sites, suggesting the possibility of post-transcriptional regulation. Consistently, reverse transcription-PCR analysis revealed that many of the changes observed upon TCERG1 knockdown were because of differences in alternative mRNA processing of the 3′-untranslated regions. Furthermore, a novel computational approach, which can identify alternatively processed events from conventional microarray data, showed that TCERG1 led to widespread alterations in mRNA processing. These findings provide the strongest support to date for a role of TCERG1 in mRNA processing and are consistent with proposals that TCERG1 couples transcription and processing. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.Fil:Muñoz, M.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Evidence for a role of the alternatively spliced ED-I sequence of fibronectin during ovarian follicular development

This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I + FN, whereas total FN levels remained relatively constant. ED-I + FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I + FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-β elicited a 2-fold stimulation of overall FN synthesis and a 4-fold increase in the synthesis of ED-I containing FN. This effect was evident at the protein (Western blots) and messenger RNA (Northern blots) levels. Although a negative correlation (P < 0.001) was detected between ED-I + FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I + FN was suggested by the observation that a recombinant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I + FN as a growth regulatory factor was further strengthened by the fact that depletion of FN from BGC-1-conditioned medium, which contained ED-I + FN, abrogated its mitogenic activity, whereas plasma FN was without effect. We propose that changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.Fil:Colman-Lerner, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Lanuza, G.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Barañao, J.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Control of alternative pre-mRNA splicing by RNA pol II elongation: Faster is not always better

The realization that the mammalian proteomic complexity is achieved with a limited number of genes demands a better understanding of alternative splicing regulation. Promoter control of alternative splicing was originally described by our group in studies performed on the fibronectin gene. Recently, other labs extended our findings to the cystic fibrosis, CD44 and CGRP genes strongly supporting a coupling between transcription and pre-mRNA splicing. A possible mechanism that would fit in these results is that the promoter itself is responsible for recruiting splicing factors, such as SR proteins, to the site of transcription, possibly through transcription factors that bind the promoter or the transcriptional enhancers. An alternative model, discussed more extensively in this review, involves modulation of RNA pol II (pol II) elongation rate. The model is supported by findings that cis- and trans-acting factors that modulate pol II elongation on a particular template also provoke changes in the alternative splicing balance of the encoded mRNAs.Fil:Nogués, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kadener, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Cramer, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:De la Mata, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Fededa, J.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Blaustein, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Srebrow, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Coordination between transcription and pre-mRNA processing

A large body of work has proved that transcription by RNA polymerase II and pre-mRNA processing are coordinated events within the cell nucleus. Capping, splicing and polyadenylation occur while transcription proceeds, suggesting that RNA polymerase II plays a role in the regulation of these events. The presence and degree of phosphorylation of the carboxy-terminal domain of RNA polymerase II large subunit is important for functioning of the capping enzymes, the assembly of spliceosomes and the binding of the cleavage/polyadenylation complex. Nuclear architecture and gene promoter structure have also been shown to play key roles in coupling between transcription and splicing. © 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.Fil:Cramer, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Srebrow, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kadener, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:De La Mata, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Melen, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Nogués, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Chromatin and alternative splicing

Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNAmediated transcriptional gene silencing. © 2010 Cold Spring Harbor Laboratory Press.Fil:Alló, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Schor, I.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Muñoz, M.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:De La Mata, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

A polar mechanism coordinates different regions of alternative splicing within a single gene

Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions. Copyright ©2005 by Elsevier Inc.Fil:Fededa, J.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Petrillo, E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kadener, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Nogués, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Pelisch, F. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Baralle, F.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Muro, A.F. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina