Biomedical subjects

Claudio R Alonso

Publications and source records attributed to Claudio R Alonso.

8 recordsLinked to original sources

PubMed2005

Nonsense-mediated RNA decay: a molecular system micromanaging individual gene activities and suppressing genomic noise.

Nonsense-mediated RNA decay (NMD) is an evolutionary conserved system of RNA surveillance that detects and degrades RNA transcripts containing nonsense mutations. Given that these mutations arise at a relatively low frequency, are there any as yet unknown substrates of NMD in a wild-type cell? With this question in mind, Mendell et al. have used a microarray assay to identify those human genes under NMD regulation. Their results show that, in human cells, NMD regulates hundreds of physiologic transcripts and not just those containing nonsense mutations. Among the NMD targets are a number of non-functional RNAs expressed from vestigial sequences derived from retroviral and transposable elements. These findings support the notion that NMD is a high profile post-transcriptional mechanism micromanaging the activity of multiple gene batteries and suppressing the expression of genetic remnants.

Animals↗

PubMed2005

The molecular elements that underlie developmental evolution.

Abundant evidence indicates that developmental evolution, the foundation of morphological evolution, is based on changes in gene function. Over the past decade a consensus has developed that transcriptional regulation, acting through enhancer sequences, is the primary level of evolutionarily significant change. Here we propose that other regulatory levels are probably as important as enhancers in developmental evolution. We also explain why these alternative regulatory levels might have been neglected, and briefly discuss ways to test our hypothesis.

Alternative Splicing↗

PubMed2004

Regulation of fibronectin splicing in sinusoidal endothelial cells from normal or injured liver.

Fn containing an extra type III domain (EIIIA in the rat, ED1 or EDA in humans) is commonly termed "fetal" fibronectin, but it is prominent during the injury response of adult tissues and mediates important early events in the response. This form is particularly apparent in acute liver injury, where it has been shown that sinusoidal endothelial cells produce EIIIA-fibronectin. This fibronectin isoform arises by alternative splicing of the primary transcript. In the present experiments, we have studied the regulation of fibronectin splicing in primary sinusoidal endothelial cells by transfecting a minigene containing the EIIIA exon and its flanking introns, driven by various promoters. The results indicate that fibronectin splicing in endothelial cells from normal liver is in part promoter-dependent. However, in cells from injured liver in which expression of both total and EIIIA-fibronectin is strikingly increased, promoter effects disappear. Because fibronectin splicing is known to be regulated in part by TGFbeta, we also examined the effect of a soluble inhibitor of the TGFbeta type 2 receptor. This agent had no effect on splicing by normal endothelial cells. By contrast, for endothelial cells from the injured liver, the splicing pattern reverted to that of normal cells, i.e., it became promoter-dependent. We conclude that, in the setting of injury in vivo, TGFbeta overrides the promoter dependence of fibronectin splicing in normal cells. The data suggest that TGFbeta modifies the spliceosome, if not through its known signaling intermediates, then through the products of genes regulated by this cytokine.

Alternative Splicing↗

PubMed2003

A Hox gene mutation that triggers nonsense-mediated RNA decay and affects alternative splicing during Drosophila development.

Nonsense mutations are usually assumed to affect protein function by generating truncated protein products. Nonetheless, it is now clear that these mutations affect not just protein synthesis but also messenger RNA stability. The surveillance mechanism responsible for the detection and degradation of 'nonsense' RNA messages is termed nonsense-mediated RNA decay (NMD). Essential biochemical components of the NMD machinery have been defined in several species. Here we identify the Drosophila orthologue of one of these factors, Upf1, and document its expression during embryogenesis. To test whether NMD acts during Drosophila development, we make use of a mutation that introduces a stop codon into a variably spliced exon of the Hox gene Ultrabithorax (Ubx). Using real-time quantitative RT-PCR we demonstrate that Ubx transcripts containing the premature stop codon are expressed at lower levels than their wild type counterpart. Unexpectedly, we also find that the same mutation significantly increases the levels of a Ubx splicing isoform that lacks the exon containing the premature termination codon. These findings indicate that NMD is operational during Drosophila development and suggest that nonsense mutations may affect development by altering the spectrum of splicing products formed, as well as by reducing or eliminating protein synthesis.

Alternative Splicing↗

PubMed2003

Drosophila miniature and dusky encode ZP proteins required for cytoskeletal reorganisation during wing morphogenesis.

We have characterised the function of two Drosophila genes, miniature and dusky, that are required for the morphological reorganisation of the apical membrane during wing epidermis differentiation. These genes encode transmembrane proteins containing a ZP (zona pellucida) domain and are homologous to several vertebrate and invertebrate apical matrix components. miniature and dusky are only expressed in tissues secreting a cuticle, and the Min protein localises to the apical membrane during the early stages of cuticle formation. We propose that Min and Dusky form a novel subfamily within the ZP domain proteins and are specifically involved in the interactions between the apical membrane, the cytoskeleton and the forming cuticle.

Amino Acid Sequence↗

PubMed2003

A slow RNA polymerase II affects alternative splicing in vivo.

Changes in promoter structure and occupation have been shown to modify the splicing pattern of several genes, evidencing a coupling between transcription and alternative splicing. It has been proposed that the promoter effect involves modulation of RNA pol II elongation rates. The C4 point mutation of the Drosophila pol II largest subunit confers on the enzyme a lower elongation rate. Here we show that expression of a human equivalent to Drosophila's C4 pol II in human cultured cells affects alternative splicing of the fibronectin EDI exon and adenovirus E1a pre-mRNA. Most importantly, resplicing of the Hox gene Ultrabithorax is stimulated in Drosophila embryos mutant for C4, which demonstrates the transcriptional control of alternative splicing on an endogenous gene. These results provide a direct proof for the elongation control of alternative splicing in vivo.

Adenoviridae↗

PubMed2002

Hox proteins: sculpting body parts by activating localized cell death.

Hox proteins shape animal structures by eliciting different developmental programs along the anteroposterior body axis. A recent study reveals that the Drosophila Hox protein Deformed directly activates the cell-death-promoting gene reaper to maintain the boundaries between distinct head segments.

Animals↗

PubMed2002

Fibronectin transcription in liver cells: promoter occupation and function in sinusoidal endothelial cells and hepatocytes.

Hepatocytes (Heps) and sinusoidal endothelial cells (SECs) perform different roles in normal and pathological liver functions through the differential expression of fibronectin (FN) polypeptides. Nonetheless, the molecular basis underlying cell-type specific FN expression remains unknown. Using liver cell isolation techniques followed by short-term primary culture and transient transfection, here, we compare the transcriptional regulation of the FN promoter in Heps and SEC in conditions that closely resemble in vivo physiology. Transfection experiments allowed us to reveal cell-type specific regulatory elements operating through the proximal regions of the FN promoter. To investigate this further, we examined the occupation patterns of key elements of the FN promoter such as the -170 CRE and -150 CCAAT sites. Transcriptional activity of mutagenised promoter constructs confirmed that in Heps, these two sites behave as a composite element critical for normal promoter activity. In addition, DNA-binding experiments demonstrate that the -170 CRE element displays a clear cell-type specific occupation with binding activities for ATF-2 and ATF-3 being specific to Heps. These results establish the starting point to investigate the molecular basis of changes in transcriptional regulation of the FN gene involved in liver pathology.

Animals↗