Biomedical subjects

Giovanni Stefani

Publications and source records attributed to Giovanni Stefani.

6 recordsLinked to original sources

PubMed2006

An RNA map predicting Nova-dependent splicing regulation.

Nova proteins are a neuron-specific alternative splicing factors. We have combined bioinformatics, biochemistry and genetics to derive an RNA map describing the rules by which Nova proteins regulate alternative splicing. This map revealed that the position of Nova binding sites (YCAY clusters) in a pre-messenger RNA determines the outcome of splicing. The map correctly predicted Nova's effect to inhibit or enhance exon inclusion, which led us to examine the relationship between the map and Nova's mechanism of action. Nova binding to an exonic YCAY cluster changed the protein complexes assembled on pre-mRNA, blocking U1 snRNP (small nuclear ribonucleoprotein) binding and exon inclusion, whereas Nova binding to an intronic YCAY cluster enhanced spliceosome assembly and exon inclusion. Assays of splicing intermediates of Nova-regulated transcripts in mouse brain revealed that Nova preferentially regulates removal of introns harbouring (or closest to) YCAY clusters. These results define a genome-wide map relating the position of a cis-acting element to its regulation by an RNA binding protein, namely that Nova binding to YCAY clusters results in a local and asymmetric action to regulate spliceosome assembly and alternative splicing in neurons.

Alternative Splicing↗

PubMed2005

Kissing complex RNAs mediate interaction between the Fragile-X mental retardation protein KH2 domain and brain polyribosomes.

Fragile-X mental retardation is caused by loss of function of a single gene encoding the Fragile-X mental retardation protein, FMRP, an RNA-binding protein that harbors two KH-type and one RGG-type RNA-binding domains. Previous studies identified intramolecular G-quartet RNAs as high-affinity targets for the RGG box, but the relationship of RNA binding to FMRP function and mental retardation remains unclear. One severely affected patient harbors a missense mutation (I304N) within the second KH domain (KH2), and some evidence suggests this domain may be involved in the proposed role of FMRP in translational regulation. We now identify the RNA target for the KH2 domain as a sequence-specific element within a complex tertiary structure termed the FMRP kissing complex. We demonstrate that the association of FMRP with brain polyribosomes is abrogated by competition with the FMRP kissing complex RNA, but not by high-affinity G-quartet RNAs. We conclude that mental retardation associated with the I304N mutation, and likely the Fragile-X syndrome more generally, may relate to a crucial role for RNAs harboring the kissing complex motif as targets for FMRP translational regulation.

Amino Acid Motifs↗

PubMed2005

Nova autoregulation reveals dual functions in neuronal splicing.

The Nova family of neuron-specific RNA-binding proteins were originally identified as targets in an autoimmune neurologic disease characterized by failure of motor inhibition. Nova-1 regulates alternative splicing of pre-mRNAs encoding the inhibitory neurotransmitter receptor subunits GABA(A)Rgamma2 and GlyRalpha2 by directly binding intronic elements, resulting in enhancement of exon inclusion. Here we identify exon E4 in the Nova-1 pre-mRNA itself, encoding a phosphorylated protein domain, as an additional target of Nova-dependent splicing regulation in the mouse spinal cord. Nova binding to E4 is necessary and sufficient for Nova-dependent exon exclusion. E4 harbors five repeats of the known Nova-binding tetranucleotide YCAY and mutation of these elements destroys Nova-dependent regulation. Furthermore, swapping of the sites from Nova-1 and GABA(A)Rgamma2 indicates that the ability of Nova to enhance or repress alternative exon inclusion is dependent on the position of the Nova-binding element within the pre-mRNA. These studies demonstrate that in addition to its previously described role as a splicing activator, Nova autoregulates its own expression by acting as a splicing repressor.

Alternative Splicing↗

PubMed2005

Emission-depth-selective auger photoelectron coincidence spectroscopy.

The collision statistics of the energy dissipation of Auger and photoelectrons emitted from an amorphized Si(100) surface is studied by measuring the Si 2p photoelectron line as well as the first plasmon loss peak in coincidence with the Si-LVV Auger transition and the associated first plasmon loss. The Si 2p plasmon intensity decreases when measured in coincidence with the Si-LVV peak. If measured in coincidence with the Si-LVV plasmon the decrease is significantly smaller. The results agree quantitatively with calculations accounting for surface, volume, and intrinsic losses as well as elastic scattering in a random medium. In this way one can determine the average emission depth of individual electrons by means of Auger photoelectron coincidence spectroscopy, which therefore constitutes a unique tool to investigate interfaces at the nanoscale level.

Journal Article↗

PubMed2004

Fragile X mental retardation protein is associated with translating polyribosomes in neuronal cells.

Fragile X mental retardation protein (FMRP) is an RNA binding protein encoded by the gene FMR1, whose expression is impaired in patients with fragile X mental retardation. The association of FMRP with polyribosomes in non-neural cell lines has previously suggested that FMRP is involved in translational regulation. However, the relevance of these studies to neuronal function has been questioned by the finding that FMRP in brain is not associated with polyribosomes, but is part of small ribonucleo-protein complexes that do not appear to include ribosomes. Here we optimize methods to analyze brain polyribosomes, allowing us to definitively demonstrate that FMRP forms complexes with cortical brain polyribosomes. Moreover, we demonstrate in neuroblastoma cells that the FMRP-polyribosome complexes are sensitive to puromycin, a drug that targets actively translating ribosomes. These data indicate that FMRP associates with functional polyribosomes in neurons.

Animals↗

PubMed2004

Characterization of Xenopus Phox2a and Phox2b defines expression domains within the embryonic nervous system and early heart field.

The closely related homeodomain containing genes, Phox2a and Phox2b, are essential for neuronal specification and differentiation within discrete subsets of neurons during vertebrate embryogenesis. We have isolated Xenopus Phox2 homologs, termed Xphox2a and Xphox2b, and characterized their expression during early development. In addition, we have characterized a Phox2a splice variant, termed Xphox2a.2, which lacks homeo- and C-terminal protein coding domains. Xphox2a, Xphox2a.2 and Xphox2b transcripts are expressed in dynamic temporal and regional patterns during nervous system development. The expression of Xphox2a and Xphox2b is only partially overlapping and includes cranial motor and interneuron populations as well as peripheral sympathetic and cranial ganglion neurons, sites linked to Phox2 expression in other species. In addition, we have identified an early domain of Xphox2a and subsequent Xphox2b expression in ventral regions of the embryo, within the developing heart field. XPhox2 expression within this domain is preceded by the gastrula-stage expression of the proneural basic helix-loop-helix transcription factor, Xash1, pointing to a new region of action for this group transcription factors during vertebrate development.

Amino Acid Sequence↗