Biomedical subjects

Irene Bozzoni

Publications and source records attributed to Irene Bozzoni.

At least 19 recordsLinked to original sources

PubMed2006

The position of yeast snoRNA-coding regions within host introns is essential for their biosynthesis and for efficient splicing of the host pre-mRNA.

Genomic location of sequences encoding small nucleolar RNAs (snoRNAs) is peculiar in all eukaryotes from yeast to mammals: most of them are encoded within the introns of host genes. In Saccharomyces cerevisiae, seven snoRNAs show this location. In this work we demonstrate that the position of snoRNA-coding regions with respect to splicing consensus sequences is critical: yeast strains expressing mutant constructs containing shorter or longer spacers (the regions between snoRNA ends and intron splice sites) show a drop in accumulation of U24 and U18 snoRNAs. Further mutational analysis demonstrates that altering the distance between the 3' end of the snoRNA and the branch point is the most important constraint for snoRNA biosynthesis, and that stable external stems, which are sometimes present in introns containing snoRNAs, can overcome the positional effect. Surprisingly enough, splicing of the host introns is clearly affected in most of these constructs indicating that, at least in S. cerevisiae, an incorrect location of snoRNA-coding sequences within the host intron is detrimental to the splicing process. This is different with respect to what was demonstrated in mammals, where the activity of the splicing machinery seems to be dominant with respect to the assembly of snoRNPs, and it is not affected by the location of snoRNA sequences. We also show that intronic box C/D snoRNA recognition and assembly of snoRNPs occur during transcription when splicing sequences are recognized.

Introns↗

PubMed2006

Preferential silencing of a common dominant rhodopsin mutation does not inhibit retinal degeneration in a transgenic model.

Autosomal dominant retinitis pigmentosa caused by the frequent rhodopsin P23H mutation is characterized by progressive photoreceptor cell death eventually leading to blindness and for which no therapies are available. Considering the gain-of-function effect exerted by the P23H mutation, strategies aimed at silencing the expression of the mutated allele, like RNA interference, are desirable. We have designed small interfering RNAs (siRNA) to silence specifically the P23H rhodopsin allele expressed by a transgenic rat model of the disease. We have selected in vitro one siRNA and generated an adeno-associated viral (AAV) vector expressing the short hairpin RNA (shRNA) based on the selected siRNA. In vitro the shRNA significantly inhibits the expression of the P23H but not the wild-type rhodopsin allele. Subretinal administration of the AAV2/5 vector encoding the shRNA in P23H transgenic rats results in inhibition of rhodopsin P23H expression that is not able to prevent or block photoreceptor degeneration. Since rhodopsin is the most abundant rod photoreceptor protein, systems resulting in more robust shRNA expression in the retina may be required to achieve therapeutic efficacy in vivo.

Alleles↗

PubMed2006

The structure of the endoribonuclease XendoU: From small nucleolar RNA processing to severe acute respiratory syndrome coronavirus replication.

Small nucleolar RNAs (snoRNAs) play a key role in eukaryotic ribosome biogenesis. In most cases, snoRNAs are encoded in introns and are released through the splicing reaction. Some snoRNAs are, instead, produced by an alternative pathway consisting of endonucleolytic processing of pre-mRNA. XendoU, the endoribonuclease responsible for this activity, is a U-specific, metal-dependent enzyme that releases products with 2'-3' cyclic phosphate termini. XendoU is broadly conserved among eukaryotes, and it is a genetic marker of nidoviruses, including the severe acute respiratory syndrome coronavirus, where it is essential for replication and transcription. We have determined by crystallography the structure of XendoU that, by refined search methodologies, appears to display a unique fold. Based on sequence conservation, mutagenesis, and docking simulations, we have identified the active site. The conserved structural determinants of this site may provide a framework for attempting to design antiviral drugs to interfere with the infectious nidovirus life cycle.

Amino Acid Sequence↗

PubMed2006

A nucleolar localizing Rev binding element inhibits HIV replication.

The Rev protein of the human immunodeficiency virus (HIV) facilitates the nuclear export of intron containing viral mRNAs allowing formation of infectious virions. Rev traffics through the nucleolus and shuttles between the nucleus and cytoplasm. Rev multimerization and interaction with the export protein CRM1 takes place in the nucleolus. To test the importance of Rev nucleolar trafficking in the HIV-1 replication cycle, we created a nucleolar localizing Rev Response Element (RRE) decoy and tested this for its anti-HIV activity. The RRE decoy provided marked inhibition of HIV-1 replication in both the CEM T-cell line and in primary CD34+ derived monocytes. These results demonstrate that titration of Rev in the nucleolus impairs HIV-1 replication and supports a functional role for Rev trafficking in this sub-cellular compartment.

Journal Article↗

PubMed2006

Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins.

XendoU is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar RNAs. It is conserved among eukaryotes and its viral homologue is essential in SARS replication and transcription. The large-scale purification and crystallization of recombinant XendoU are reported. The tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (EDTA, imidazole): aggregation is a potential drawback when purifying and crystallizing His-tagged proteins, which are widely used, especially in high-throughput structural studies. Purified monodisperse XendoU crystallized in two different space groups: trigonal P3(1)21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution.

Animals↗

PubMed2006

Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model.

Duchenne muscular dystrophy is an X-linked muscle disease characterized by mutations in the dystrophin gene. Many of these can be corrected at the posttranscriptional level by skipping the mutated exon. We have obtained persistent exon skipping in mdx mice by tail vein injection with an adeno-associated viral (AAV) vector expressing antisense sequences as part of the stable cellular U1 small nuclear RNA. Systemic delivery of the AAV construct resulted in effective body-wide colonization, significant recovery of the functional properties in vivo, and lower creatine kinase serum levels, suggesting an overall decrease in muscle wasting. The transduced muscles rescued dystrophin expression and displayed a significant recovery of function toward the normal values at single muscle fiber level. This approach provides solid bases for a systemic use of AAV-mediated antisense-U1 small nuclear RNA expression for the therapeutic treatment of Duchenne muscular dystrophy.

Animals↗

PubMed2006

Chimeric adeno-associated virus/antisense U1 small nuclear RNA effectively rescues dystrophin synthesis and muscle function by local treatment of mdx mice.

Duchenne muscular dystrophy (DMD) is a X-linked myopathy in which deletions and point mutations in the dystrophin gene abolish dystrophin expression. The defect can often be corrected at the posttranscriptional level by exon skipping. In an animal model of DMD, the mdx mouse, a point mutation in exon 23 of the dystrophin gene introduces a premature stop codon. Skipping of this exon reestablishes the open reading frame in the dystrophin mRNA. We have obtained persistent exon skipping in mdx mice by local muscle injection of AAV vectors expressing antisense sequences fused to either U1 or U7 small nuclear RNA (snRNA). In the transduced muscles, dystrophin expression, amelioration of muscle morphology, and significant force recovery were obtained. These data indicate that the expression of antisense snRNAs, combined with their efficient muscular delivery through AAV vectors, is a powerful strategy for the therapeutic treatment of DMD. Like U7 snRNA, spliceosomal U1 snRNA is also a suitable backbone for the expression of antisense molecules active in exon skipping.

Animals↗

PubMed2005

A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis.

MicroRNAs play important roles in cell differentiation by acting as translational inhibitors of specific target genes. Here we show that human granulocytic differentiation is controlled by a regulatory circuitry involving miR-223 and two transcriptional factors, NFI-A and C/EBPalpha. The two factors compete for binding to the miR-223 promoter: NFI-A maintains miR-223 at low levels, whereas its replacement by C/EBPalpha, following retinoic acid (RA)-induced differentiation, upregulates miR-223 expression. The competition by C/EBPalpha and the granulocytic differentiation are favored by a negative-feedback loop in which miR-223 represses NFI-A translation. In line with this, both RNAi against NFI-A and ectopic expression of miR-223 in acute promyelocytic leukemia (APL) cells enhance differentiation, whereas miR-223 knockdown inhibits the differentiation response to RA. Altogether, our data indicate that miR-223 plays a crucial role during granulopoiesis and point to the NFI-A repression as an important molecular pathway mediating gene reprogramming in this cell lineage.

Binding Sites↗

PubMed2005

A loxP-containing pol II promoter for RNA interference is reversibly regulated by Cre recombinase.

Several DNA vectors for RNA interference in mammalian cells have been described. These express a short hairpin RNA (shRNA) that is subsequently processed into mature small interfering RNAs (siRNAs). We previously developed the siRNA-expressing vector psiUx based on the polII promoter of the U1 small nuclear RNA gene. Here we describe the conversion of such construct into an inducible system. The starting construct psiUStuff contains a loxP-Stuffer-loxP cassette just upstream the transcription initiation site and does not express the shRNA until the two canonical loxP sites undergo Cre-mediated recombination. If sustained expression of the recombinase is maintained, transcription is repressed and shRNA synthesis is abolished. Therefore, in our system the Cre recombinase exhibits the dual function of activator and repressor allowing the on/off regulation of siRNAs production. Using a Cre recombinase whose transcription is under the control of a tetOn system, we show the temporally controlled expression of an shRNA directed towards the lamin A/C mRNA, as well as the regulated knockdown of its target.

DNA Polymerase II↗

PubMed2005

Functional characterization of XendoU, the endoribonuclease involved in small nucleolar RNA biosynthesis.

XendoU is the endoribonuclease involved in the biosynthesis of a specific subclass of Xenopus laevis intron-encoded small nucleolar RNAs. XendoU has no homology to any known cellular RNase, although it has sequence similarity with proteins tentatively annotated as serine proteases. It has been recently shown that XendoU represents the cellular counterpart of a nidovirus replicative endoribonuclease (NendoU), which plays a critical role in viral replication and transcription. In this paper, we combined prediction and experimental data to define the amino acid residues directly involved in XendoU catalysis. Specifically, we find that XendoU residues Glu-161, Glu-167, His-162, His-178, and Lys-224 are essential for RNA cleavage, which occurs in the presence of manganese ions. Furthermore, we identified the RNA sequence required for XendoU binding and showed that the formation of XendoU-RNA complex is Mn2+-independent.

Amino Acid Sequence↗

PubMed2005

The cotranscriptional assembly of snoRNPs controls the biosynthesis of H/ACA snoRNAs in Saccharomyces cerevisiae.

The carboxy-terminal domain (CTD) of RNA polymerase II large subunit acts as a platform to assemble the RNA processing machinery in a controlled way throughout the transcription cycle. In yeast, recent findings revealed a physical connection between phospho-CTD, generated by the Ctk1p kinase, and protein factors having a function in small nucleolar RNA (snoRNA) biogenesis. The snoRNAs represent a large family of polymerase II noncoding transcripts that are associated with highly conserved polypeptides to form stable ribonucleoprotein particles (snoRNPs). In this work, we have studied the biogenesis of the snoRNPs belonging to the box H/ACA class. We report that the assembly factor Naf1p and the core components Cbf5p and Nhp2p are recruited on H/ACA snoRNA genes very early during transcription. We also show that the cotranscriptional recruitment of Naf1p and Cbf5p is Ctk1p dependent and that Ctk1p and Cbf5p are required for preventing the readthrough into the snoRNA downstream genes. All these data suggest that proper cotranscriptional snoRNP assembly controls 3'-end formation of snoRNAs and, consequently, the release of a functional particle.

Chromatin Immunoprecipitation↗

PubMed2005

Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation.

In eukaryotes ribosome biogenesis required that rRNAs primary transcripts are assembled in pre-ribosomal particles and processed. Protein factors and pre-ribosomal complexes involved in this complex pathway are not completely depicted. The essential ORF YPR143W encodes in yeast for an uncharacterized protein product, named here Rrp15p. Cellular function of Rrp15p has not so far defined even if nucleolar location was referred. With the aim to define the possible role of this orphan gene, we performed TAP-tagging of Rrp15p and investigated its molecular association with known pre-ribosomal complexes. Comparative sucrose gradient sedimentation analyses of yeast lysates expressing the TAP-tagged Rrp15p, strongly indicated that this protein is a component of the pre-60S particles. Northern hybridization, primer extension and functional proteomics on TAP-affinity isolated complexes proved that Rrp15p predominately associated with pre-rRNAs and proteins previously characterized as components of early pre-60S ribosomal particles. Finally, depletion of Rrp15p inhibited the accumulation of 27S and 7S pre-rRNAs and 5.8S and 25S mature rRNA. These results provide the first indication that Rrp15p is a novel factor involved in the early maturation steps of the 60S subunits. Moreover, the identification of the protein kinase CK2 in the Rrp15p-containing pre-ribosomal particles here reported, sustains the link between ribosome synthesis and cell cycle progression.

Amino Acid Sequence↗

PubMed2004

TOP promoter elements control the relative ratio of intron-encoded snoRNA versus spliced mRNA biosynthesis.

In vertebrates almost all snoRNAs are encoded in introns of a specific subclass of polII transcripts: the TOP genes. The majority of these RNAs originate through debranching of the spliced introns, the rest through endonucleolytic cleavage of the precursor that contains them. In both cases it has been suggested that snoRNP factors associate at early steps during transcription and control snoRNA biogenesis. Here, we analyzed the specific case of the U16 snoRNA that was shown to originate mainly through endonucleolytic cleavage. We show that TOP promoter elements determine a specific ratio of snoRNA and mRNA production. Under the control of these sequences the snoRNA is likely to originate from both splicing and cleavage of the pre-mRNA. Conversely, canonical polII promoter elements seem not to be compatible with snoRNA release through the cleavage reaction and produce a lower snoRNA/mRNA ratio. In addition, we show that the proximal part of the TOP promoter is responsible for this peculiar post-transcriptional process that controls the relative ratio between snoRNA and mRNA.

Animals↗

PubMed2004

Coupling between snoRNP assembly and 3' processing controls box C/D snoRNA biosynthesis in yeast.

RNA polymerase II transcribes genes encoding proteins and a large number of small stable RNAs. While pre-mRNA 3'-end formation requires a machinery ensuring tight coupling between cleavage and polyadenylation, small RNAs utilize polyadenylation-independent pathways. In yeast, specific factors required for snRNA and snoRNA 3'-end formation were characterized as components of the APT complex that is associated with the core complex of the cleavage/polyadenylation machinery (core-CPF). Other essential factors were identified as independent components: Nrd1p, Nab3p and Sen1p. Here we report that mutations in the conserved box D of snoRNAs and in the snoRNP-specific factor Nop1p interfere with transcription and 3'-end formation of box C/D snoRNAs. We demonstrate that Nop1p is associated with box C/D snoRNA genes and that it interacts with APT components. These data suggest a mechanism of quality control in which efficient transcription and 3'-end formation occur only when nascent snoRNAs are successfully assembled into functional particles.

Base Sequence↗

PubMed2004

Msx2 and necdin combined activities are required for smooth muscle differentiation in mesoangioblast stem cells.

Little is known about the molecular mechanism underlying specification and differentiation of smooth muscle (SM), and this is, at least in part, because of the few cellular systems available to study the acquisition of a SM phenotype in vitro. Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm, including SM. We performed a DNA microarray analysis of a mesoangioblast clone that spontaneously expresses an immature SM phenotype and compared it with a sister clone mainly composed of undifferentiated progenitor cells. This study allowed us to define a gene expression profile for "stem" cells versus smooth muscle cells (SMCs) in the absence of differentiation inducers such as transforming growth factor beta. Two transcription factors, msx2 and necdin, are expressed at least 100 times more in SMCs than in stem cells, are coexpressed in all SMCs and tissues, are induced by transforming growth factor beta, and, when coexpressed, induce a number of SM markers in mesoangioblast, fibroblast, and endothelial cell lines. Conversely, their downregulation through RNA interference results in a decreased expression of SM markers. These data support the hypothesis that Msx2 and necdin act as master genes regulating SM differentiation in at least a subset of SMCs.

Animals↗

PubMed2004

A new vector, based on the PolII promoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells.

Several vectors for the induction of RNA interference in mammalian cells have been described,based mainly on polIII-dependent promoters. They transcribe short hairpin RNAs (shRNA) that,after being processed into short interfering RNAs (siRNAs), mediate the degradation of the target mRNA. Here, we describe the construction of a new siRNA-expressing vector (psiUx) based on the strong and ubiquitous polII-dependent promoter of the human U1 small nuclear RNA (snRNA)gene. In psiUx, the only constraint for the shRNA sequence is a purine at position +1, since specific 3'-end formation is achieved by a box element located downstream of the transcribed region. Several constructs were designed against the lamin A/C target. Depending on the structure of the shRNA transcribed, a preferential or exclusive accumulation of the antisense strand is obtained, thus avoiding possible nonspecific targeting by the sense strand. In all cases tested, very effective siRNAs were produced, thus providing a proof-of-principle that a snRNA-type polII promoter can be used for the expression of siRNAs. We show that psiUx ensures high levels of expression and efficient knock down of the target gene also in stable cell lines.

Genetic Vectors↗

PubMed2003

Purification, cloning, and characterization of XendoU, a novel endoribonuclease involved in processing of intron-encoded small nucleolar RNAs in Xenopus laevis.

Here we report the purification, from Xenopus laevis oocyte nuclear extracts, of a new endoribonuclease, XendoU, that is involved in the processing of the intron-encoded box C/D U16 small nucleolar RNA (snoRNA) from its host pre-mRNA. Such an activity has never been reported before and has several uncommon features that make it quite a novel enzyme: it is poly(U)-specific, it requires Mn(2+) ions, and it produces molecules with 2'-3'-cyclic phosphate termini. Even if XendoU cleaves U-stretches, it displays some preferential cleavage on snoRNA precursor molecules. XendoU also participates in the biosynthesis of another intron-encoded snoRNA, U86, which is contained in the NOP56 gene of Xenopus laevis. A common feature of these snoRNAs is that their production is alternative to that of the mRNA, suggesting an important regulatory role for all the factors involved in the processing reaction.

Amino Acid Sequence↗

PubMed2003

Cic1p/Nsa3p is required for synthesis and nuclear export of 60S ribosomal subunits.

Cic1p/Nsa3p was previously reported to be associated with the 26S proteasome and required for the degradation of specific substrates, but was also shown to be associated with early pre-60S particles and to be localized to the nucleolus. Here we report that Cic1p/Nsa3p is required for the synthesis of 60S ribosome subunits. A temperature-sensitive lethal cic1-2 point mutation inhibits synthesis of the mature 5.8S and 25S rRNAs. Release of the pre-60S particles from the nucleolus to the nucleoplasm was also inhibited as judged by the nuclear accumulation of an Rpl11b-GFP reporter construct. We suggest that Cic1p/Nsa3p associates early with nascent preribosomal particles and is required for correct processing and nuclear release of large ribosomal subunit precursors.

Base Sequence↗