The interplay between the master transcription factor PU.1 and miR-424 regulates human monocyte/macrophage differentiation.

We describe a pathway by which the master transcription factor PU.1 regulates human monocyte/macrophage differentiation. This includes miR-424 and the transcriptional factor NFI-A. We show that PU.1 and these two components are interlinked in a finely tuned temporal and regulatory circuitry: PU.1 activates the transcription of miR-424, and this up-regulation is involved in stimulating monocyte differentiation through miR-424-dependent translational repression of NFI-A. In turn, the decrease in NFI-A levels is important for the activation of differentiation-specific genes such as M-CSFr. In line with these data, both RNAi against NFI-A and ectopic expression of miR-424 in precursor cells enhance monocytic differentiation, whereas the ectopic expression of NFI-A has an opposite effect. The interplay among these three components was demonstrated in myeloid cell lines as well as in human CD34+ differentiation. These data point to the important role of miR-424 and NFI-A in controlling the monocyte/macrophage differentiation program.

Expression of ribosomal protein genes and regulation of ribosome biosynthesis in Xenopus development.

Studies on ribosome biosynthesis in developing Xenopus oocytes and embryos, and after microinjection of cloned ribosomal-protein genes, have revealed that the synthesis of ribosomal proteins (r-proteins) is controlled by two types of regulation: (1) a post-transcriptional regulation, operated by feedback of the r-proteins themselves, controls processing and stability of r-protein transcripts and thus the amount of the corresponding mRNA present in the cell; and (2) a translational regulation controls the efficiency of utilization of r-protein mRNA (rp-mRNA) in response to the cellular needs for new ribosomes.

Emerging role for microRNAs in acute promyelocytic leukemia.

Hematopoiesis is highly controlled by lineage-specific transcription factors that, by interacting with specific DNA sequences, directly activate or repress specific gene expression. These transcription factors have been found mutated or altered by chromosomal translocations associated with leukemias, indicating their role in the pathogenesis of these malignancies. The post-genomic era, however, has shown that transcription factors are not the only key regulators of gene expression. Epigenetic mechanisms such as DNA methylation, posttranslational modifications of histones, remodeling of nucleosomes, and expression of small regulatory RNAs all contribute to the regulation of gene expression and determination of cell and tissue specificity. Deregulation ofthese epigenetic mechanisms cooperates with genetic alterations to the establishment and progression of tumors. MicroRNAs (miRNAs) are negative regulators of the expression of genes involved in development, differentiation, proliferation, and apoptosis. Their expression appears to be tissue-specific and highly regulated according to the cell's developmental lineage and stage. Interestingly, miRNAs expressed in hematopoietic cells have been found mutated or altered by chromosomal translocations associated with leukemias. The expression levels of a specific miR-223 correlate with the differentiation fate of myeloid precursors. The activation of both pathways of transcriptional regulation by the myeloid lineage-specific transcription factor C/EBPalpha (CCAAT/enhancer-binding protein-alpha), and posttranscriptional regulation by miR-223 appears essential for granulocytic differentiation and clinical response of acute promyelocytic leukemia (APL) blasts to all-trans retinoic acid (ATRA). Together, this evidence underlies transcription factors, chromatin remodeling, and miRNAs as ultimate determinants for the correct organization of cell type-specific gene arrays and hematopoietic differentiation, therefore providing new targets for the diagnosis and treatment of leukemias.

Identification of a novel element required for processing of intron-encoded box C/D small nucleolar RNAs in Saccharomyces cerevisiae.

Processing of intron-encoded box C/D small nucleolar RNAs (snoRNAs) in metazoans through both the splicing-dependent and -independent pathways requires the conserved core motif formed by boxes C and D and the adjoining 5'-3'-terminal stem. By comparative analysis, we found that five out of six intron-encoded box C/D snoRNAs in yeast do not possess a canonical terminal stem. Instead, complementary regions within the flanking host intron sequences have been identified in all these cases. Here we show that these sequences are essential for processing of U18 and snR38 snoRNAs and that they compensate for the lack of a canonical terminal stem. We also show that the Rnt1p endonuclease, previously shown to be required for the processing of many snoRNAs encoded by monocistronic or polycistronic transcriptional units, is not required for U18 processing. Our results suggest a role of the complementary sequences in the early recognition of intronic snoRNA substrates and point out the importance of base pairing in favoring the communication between boxes C and D at the level of pre-snoRNA molecules for efficient assembly with snoRNP-specific factors.

RNA-protein interactions in the nuclei of Xenopus oocytes: complex formation and processing activity on the regulatory intron of ribosomal protein gene L1.

The gene encoding ribosomal protein L1 in Xenopus laevis is known to be posttranscriptionally regulated; the third intron can be processed from the pre-mRNA in two alternative ways, resulting either in the production of L1 mRNA or in the release of a small nucleolar RNA (U16). The formation of splicing complexes was studied in vivo by oocyte microinjection. We show that spliceosome assembly is impaired on the L1 third intron and that the low efficiency of the process is due to the presence of suboptimal consensus sequences. An analysis of heterogeneous nuclear ribonucleoprotein (hnRNP) distribution was also performed, revealing a distinct site for hnRNP C binding proximal to the 5' end of the L1 third intron. Cleavage, leading to the production of the small nucleolar RNA U16, occurs in the same position, and we show that conditions under which hnRNP C binding is reduced result in an increase of the processing activity of the intron.

Processing of the intron-encoded U18 small nucleolar RNA in the yeast Saccharomyces cerevisiae relies on both exo- and endonucleolytic activities.

Many small nucleolar RNAs (snoRNAs) are encoded within introns of protein-encoding genes and are released by processing of their host pre-mRNA. We have investigated the mechanism of processing of the yeast U18 snoRNA, which is found in the intron of the gene coding for translational elongation factor EF-1beta. We have focused our analysis on the relationship between splicing of the EF-1beta pre-mRNA and production of the mature snoRNA. Mutations inhibiting splicing of the EF-1beta pre-mRNA have been shown to produce normal U18 snoRNA levels together with the accumulation of intermediates deriving from the pre-mRNA, thus indicating that the precursor is an efficient processing substrate. Inhibition of 5'-->3' exonucleases obtained by insertion of G cassettes or by the use of a rat1-1 xrn1Delta mutant strain does not impair U18 release. In the Exo- strain, 3' cutoff products, diagnostic of an endonuclease-mediated processing pathway, were detected. Our data indicate that biosynthesis of the yeast U18 snoRNA relies on two different pathways, depending on both exonucleolytic and endonucleolytic activities: a major processing pathway based on conversion of the debranched intron and a minor one acting by endonucleolytic cleavage of the pre-mRNA.

Identification of the sequences responsible for the splicing phenotype of the regulatory intron of the L1 ribosomal protein gene of Xenopus laevis.

Splicing of the regulated third intron of the L1 ribosomal protein gene of Xenopus laevis has been studied in vivo by oocyte microinjection of wild-type and mutant SP6 precursor RNAs and in vitro in the heterologous HeLa nuclear extract. We show that two different phenomena combine to produce the peculiar splicing phenotype of this intron. One, which can be defined constitutive, shows the same features in the two systems and leads to the accumulation of spliced mRNA, but in very small amounts. The low efficiency of splicing is due to the presence of a noncanonical 5' splice site which acts in conjunction with sequences present in the 3' portion of the intron. The second leads to the massive conversion of the pre-mRNA into site specific truncated molecules. This has the effect of decreasing the concentration of the pre-mRNA available for splicing. We show that this aberrant cleavage activity occurs only in the in vivo oocyte system and depends on the presence of an intact U1 RNA.

In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis.

It was recently shown that a new class of small nuclear RNAs is encoded in introns of protein-coding genes and that they originate by processing of the pre-mRNA in which they are contained. Little is known about the mechanism and the factors involved in this new type of processing. The L1 ribosomal protein gene of Xenopus laevis is a well-suited system for studying this phenomenon: several different introns encode for two small nucleolar RNAs (snoRNAs; U16 and U18). In this paper, we analyzed the in vitro processing of these snoRNAs and showed that both are released from the pre-mRNA by a common mechanism: endonucleolytic cleavages convert the pre-mRNA into a precursor snoRNA with 5' and 3' trailer sequences. Subsequently, trimming converts the pre-snoRNAs into mature molecules. Oocyte and HeLa nuclear extracts are able to process X. laevis and human substrates in a similar manner, indicating that the processing of this class of snoRNAs relies on a common and evolutionarily conserved mechanism. In addition, we found that the cleavage activity is strongly enhanced in the presence of Mn2+ ions.

Gene dosage alteration of L2 ribosomal protein genes in Saccharomyces cerevisiae: effects on ribosome synthesis.

In Saccharomyces cerevisiae, the genes coding for the ribosomal protein L2 are present in two copies per haploid genome. The two copies, which encode proteins differing in only a few amino acids, contribute unequally to the L2 mRNA pool: the L2A copy makes 72% of the mRNA, while the L2B copy makes only 28%. Disruption of the L2B gene (delta B strain) did not lead to any phenotypic alteration, whereas the inactivation of the L2A copy (delta A strain) produced a slow-growth phenotype associated with decreased accumulation of 60S subunits and ribosomes. No intergenic compensation occurred at the transcriptional level in the disrupted strains; in fact, delta A strains contained reduced levels of L2 mRNA, whereas delta B strains had almost normal levels. The wild-type phenotype was restored in the delta A strains by transformation with extra copies of the intact L2A or L2B gene. As already shown for other duplicated genes (Kim and Warner, J. Mol. Biol. 165:79-89, 1983; Leeret al., Curr. Genet. 9:273-277, 1985), the difference in expression of the two gene copies could be accounted for via differential transcription activity. Sequence comparison of the rpL2 promoter regions has shown the presence of canonical HOMOL1 boxes which are slightly different in the two genes.

In vivo identification of nuclear factors interacting with the conserved elements of box C/D small nucleolar RNAs.

The U16 small nucleolar RNA (snoRNA) is encoded by the third intron of the L1 (L4, according to the novel nomenclature) ribosomal protein gene of Xenopus laevis and originates from processing of the pre-mRNA in which it resides. The U16 snoRNA belongs to the box C/D snoRNA family, whose members are known to assemble in ribonucleoprotein particles (snoRNPs) containing the protein fibrillarin. We have utilized U16 snoRNA in order to characterize the factors that interact with the conserved elements common to the other members of the box C/D class. In this study, we have analyzed the in vivo assembly of U16 snoRNP particles in X. laevis oocytes and identified the proteins which interact with the RNA by label transfer after UV cross-linking. This analysis revealed two proteins, of 40- and 68-kDa apparent molecular size, which require intact boxes C and D together with the conserved 5',3'-terminal stem for binding. Immunoprecipitation experiments showed that the p40 protein corresponds to fibrillarin, indicating that this protein is intimately associated with the RNA. We propose that fibrillarin and p68 represent the RNA-binding factors common to box C/D snoRNPs and that both proteins are essential for the assembly of snoRNP particles and the stabilization of the snoRNA.

The ABF1 factor is the transcriptional activator of the L2 ribosomal protein genes in Saccharomyces cerevisiae.

The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae. In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies. The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.

Multifunctional System-on-Glass for Lab-on-Chip applications.

Lab-on-Chip are miniaturized systems able to perform biomolecular analysis in shorter time and with lower reagent consumption than a standard laboratory. Their miniaturization interferes with the multiple functions that the biochemical procedures require. In order to address this issue, our paper presents, for the first time, the integration on a single glass substrate of different thin film technologies in order to develop a multifunctional platform suitable for on-chip thermal treatments and on-chip detection of biomolecules. The proposed System on-Glass hosts thin metal films acting as heating sources; hydrogenated amorphous silicon diodes acting both as temperature sensors to monitor the temperature distribution and photosensors for the on-chip detection and a ground plane ensuring that the heater operation does not affect the photodiode currents. The sequence of the technological steps, the deposition temperatures of the thin films and the parameters of the photolithographic processes have been optimized in order to overcome all the issues of the technological integration. The device has been designed, fabricated and tested for the implementation of DNA amplification through the Polymerase Chain Reaction (PCR) with thermal cycling among three different temperatures on a single site. The glass has been connected to an electronic system that drives the heaters and controls the temperature and light sensors. It has been optically and thermally coupled with another glass hosting a microfluidic network made in polydimethylsiloxane that includes thermally actuated microvalves and a PCR process chamber. The successful DNA amplification has been verified off-chip by using a standard fluorometer.

The lack of the Celf2a splicing factor converts a Duchenne genotype into a Becker phenotype.

Substitutions, deletions and duplications in the dystrophin gene lead to either the severe Duchenne muscular dystrophy (DMD) or mild Becker muscular dystrophy depending on whether out-of-frame or in-frame transcripts are produced. We identified a DMD case (GSΔ44) where the correlation between genotype and phenotype is not respected, even if carrying a typical Duchenne mutation (exon 44 deletion) a Becker-like phenotype was observed. Here we report that in this patient, partial restoration of an in-frame transcript occurs by natural skipping of exon 45 and that this is due to the lack of Celf2a, a splicing factor that interacts with exon 45 in the dystrophin pre-mRNA. Several experiments are presented that demonstrate the central role of Celf2a in controlling exon 45 splicing; our data point to this factor as a potential target for the improvement of those DMD therapeutic treatments, which requires exon 45 skipping.

microRNAs Modulate Spatial Memory in the Hippocampus and in the Ventral Striatum in a Region-Specific Manner.

MicroRNAs are endogenous, noncoding RNAs crucial for the post-transcriptional regulation of gene expression. Their role in spatial memory formation, however, is poorly explored. In this study, we analyzed learning-induced microRNA expression in the hippocampus and in the ventral striatum. Among miRNAs specifically downregulated by spatial training, we focused on the hippocampus-specific miR-324-5p and the ventral striatum-specific miR-24. In vivo overexpression of the two miRNAs demonstrated that miR-324-5p is able to impair memory if administered in the hippocampus but not in the ventral striatum, while the opposite is true for miR-24. Overall, these findings demonstrate a causal relationship between miRNA expression changes and spatial memory formation. Furthermore, they provide support for a regional dissociation in the post-transcriptional processes underlying spatial memory in the two brain structures analyzed.

Clustered and interspersed repetitive DNA sequences in four amphibian species with different genome size.

We have compared the amount of clustered and interspersed repetitive sequences in the genome of four Amphibia with different DNA contents per haploid nucleus: two Anura (Xenopus laevis, 3 pg and Bufo bufo, 7 pg) and two Urodela (Triturus cristatus, 23 pg and Necturus maculosus, 52 pg). High molecular weight DNA of the four species was denatured and reassociated to the same Cot in order to obtain duplex sequences with a similar reiteration frequency. Single-stranded DNA was digested off with the Aspergillus S1 nuclease. DNA was then fractionated according to the molecular weight through an agarose A-50 column. We found that the amount of long repetitive sequences is roughly proportional to the genome size in the four species, while the number of short (about 300 base pairs) repetitive sequences is increased many-fold in the species with the larger DNA content, both in Anura and in Urodela.

Splicing of Xenopus laevis ribosomal protein RNAs is inhibited in vivo by antisera to ribonucleoproteins containing U1 small nuclear RNA.

The activity of antisera against ribonucleoproteins containing U1 small nuclear RNA (Sm and RNP) has been analysed on pol II transcripts in an in vivo system. Xenopus laevis ribosomal protein gene transcripts are accumulated in the form of precursor RNA when either of the two kinds of antisera are injected into the germinal vesicles of X. laevis oocytes before the injection of purified L1 and L14 ribosomal protein genes. No effect on the accumulation of mature histone mRNA is detected when X. laevis histone genes are injected together with the RNP antiserum. These results strongly suggest that U1-RNP complexes play an essential role in intron removal in vivo.

Expression of two Xenopus laevis ribosomal protein genes in injected frog oocytes. A specific splicing block interferes with the L1 RNA maturation.

The expression of two Xenopus laevis ribosomal protein genes (L1 and L14) has been analysed by microinjection of the cloned genomic sequences into frog oocyte nuclei. While the injection of the L14 gene causes the accumulation of the corresponding protein in large excess with respect to that synthesized endogenously, the L1 gene does not. Analysis of the RNA shows that both genes are actively transcribed. The seven-intron-containing L14 transcript is completely processed to a mature form, while two out of nine intron sequences persist in the L1 transcript. This precursor RNA is confined to the nucleus; its accumulation is due to a specific block of splicing operating at the level of two defined introns and not to saturation of the processing apparatus of the oocyte. The different behaviour of the two genes may reflect different mechanisms of regulation which, in the case of the L1 gene, could operate at the level of splicing.

Pur-alpha functionally interacts with FUS carrying ALS-associated mutations.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to motor neuron loss. Fused in sarcoma (FUS) protein carrying ALS-associated mutations localizes to stress granules and causes their coalescence into larger aggregates. Here we show that Pur-alpha physically interacts with mutated FUS in an RNA-dependent manner. Pur-alpha colocalizes with FUS carrying mutations in stress granules of motoneuronal cells differentiated from induced pluripotent stem cells and that are derived from ALS patients. We observe that both Pur-alpha and mutated FUS upregulate phosphorylation of the translation initiation factor eukaryotic translation initiation factor 2 alpha and consistently inhibit global protein synthesis. In vivo expression of Pur-alpha in different Drosophila tissues significatively exacerbates the neurodegeneration caused by mutated FUS. Conversely, the downregulation of Pur-alpha in neurons expressing mutated FUS significatively improves fly climbing activity. All these findings suggest that Pur-alpha, through the control of mRNA translation, might be involved in the pathogenesis of ALS associated with the mutation of FUS, and that an alteration of protein synthesis may be directly implicated in the disease. Finally, in vivo RNAi-mediated ablation of Pur-alpha produced locomotion defects in Drosophila, indicating a pivotal role for this protein in the motoneuronal function.

Inhibition of human immunodeficiency virus type 1 replication by nuclear chimeric anti-HIV ribozymes in a human T lymphoblastoid cell line.

Human immunodeficiency virus (HIV) infection represents one of the most challenging systems for gene therapy. Thanks to the extended knowledge of the molecular biology of the HIV life cycle, many different strategies have been developed including transdominant modifications of HIV proteins, RNA decoys, antisense RNA, ribozymes, and intracellular antibody fragments. In this paper, we have tested in a human T lymphoblastoid cell line the antiviral activity of ribozymes specifically designed to co-localize inside the nucleus with the Rev pre-mRNA before it is spliced and transported to the cytoplasm. This result was obtained by inserting the ribozyme in the spliceosomal U1 small nuclear RNA (snRNA) and in a derivative that has perfect complementarity with the 5' splice site of the Rev pre-mRNA. These ribozymes were tested in human T cell clones and were shown to be very efficient in inhibiting viral replication. Not only were the p24 levels in the culture medium drastically reduced but so were the intracellular HIV transcripts. Control disabled ribozymes enabled us to show the specificity of the ribozyme activity. Therefore, these constructs have potential utility for gene therapy of HIV-1 infection.

Complementarity of conserved sequence elements present in 28S ribosomal RNA and in ribosomal protein genes of Xenopus laevis and Xenopus tropicalis.

The sequence analysis of the L1 ribosomal protein (r-protein) gene of Xenopus laevis has revealed a strong homology in four out of the nine introns of the gene; this homology region spans 60 nucleotides (nt) with 80% homology [Loreni et al., EMBO J. 4 (1985) 3483-3488]. We have extended our analysis to X. tropicalis, a species which is closely related to X. laevis. Partial sequencing of the isolated L1 gene has revealed that these 60-nt homology regions are also present in at least two introns of the X. tropicalis L1 gene. Computer analysis has revealed that perfect nt sequence complementarity exists between 13 nt of this intron region and the 28S ribosomal RNA in a region which is conserved in all eukaryotes, suggesting a possible base-pairing interaction between these two sequences.