MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells.

A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protein.

Expression of the HMGI(Y) gene products in human neuroblastic tumours correlates with differentiation status.

HMGI and HMGY are splicing variants of the HMGI(Y) gene and together with HMGI-C, belong to a family of DNA binding proteins involved in maintaining active chromatin conformation and in the regulation of gene transcription. The expression of the HMGI(Y) gene is maximal during embryonic development, declines in adult differentiated tissues and is reactivated in most transformed cells in vitro and in many human cancers in vivo. The HMGI(Y) genomic locus is frequently rearranged in mesenchymal tumours, suggesting a biological role for HMGI(Y) gene products in tumour biology. HMGIs are both target and modulators of retinoic acid activity. In fact, HMGI(Y) gene expression is differentially regulated by retinoic acid in retinoid-sensitive and -resistant neuroblastoma cells, while HMGI-C participates in conferring retinoic acid resistance in some neuroblastoma cells. In this paper we show that HMGI and HMGY isoforms are equally regulated by retinoic acid in neuroblastoma cell lines at both RNA and protein levels. More importantly our immunohistochemical analysis shows that, although HMGI(Y) is expressed in all neuroblastic tumours, consistently higher levels are observed in less differentiated neuroblastomas compared to more differentiated ganglioneuromas, indicating that HMGI(Y) expression should be evaluated as a potential diagnostic and prognostic marker in neuroblastic tumours.

cDNA cloning and developmental expression of cellular nucleic acid-binding protein (CNBP) gene in Xenopus laevis.

The cloning and sequencing of a cDNA corresponding to one of the two Xenopus cellular nucleic acid binding protein (CNBP) genes are presented. Comparison of this cDNA sequence (xCNBP2) with the other previously reported (xCNBP1) reveals that, while the cDNA sequences are somewhat divergent, the amino acid sequences are mostly unchanged. It has been determined that both gene copies can generate a shorter transcript, likely due to alternative splicing, as previously demonstrated in human cells. The comparison of the cDNA sequences of Xenopus and of other species shows that the missing cDNA tract of Xenopus does not coincide with the others, consistent with the utilization of different splicing donor sites. The two gene copies are expressed at comparable levels, since the two corresponding mRNAs are similarly represented both in oocyte and embryo poly(A)(+) RNA. However, the shorter CNBP transcripts are slightly less represented than the longer CNBP transcripts, in both the oocyte and embryo. CNBP mRNA accumulation during development decreases before the mid-blastula stage and increases again thereafter. The polysome association of CNBP mRNA and the binding activity of CNBP to its target sequence of ribosomal protein mRNA 5'UTR have been analysed during development.

Resistance of ribosomal protein mRNA translation to protein synthesis shutoff induced by poliovirus.

Poliovirus infection induces an overall inhibition of host protein synthesis, although some mRNAs continue to be translated, suggesting different translation requirements for cellular mRNAs. It is known that ribosomal protein mRNAs are translationally regulated and that the phosphorylation of ribosomal protein S6 is involved in the regulation. Here, we report that the translation of ribosomal protein mRNAs resists poliovirus infection and correlates with an increase in p70(s6k) activity and phosphorylation of ribosomal protein S6.

Small nucleolar RNAs and nucleolar proteins in Xenopus anucleolate embryos.

We investigated the presence and localization, in the cells of anucleolate mutant embryos of Xenopus laevis, of three representative small nucleolar RNAs (snoRNAs), U3, U15 and U17, and of two nucleolar proteins, nucleolin and fibrillarin. The levels of the three snoRNAs in the anucleolate mutant are the same as in normal embryos, in contrast to 5S RNA and ribosomal proteins. In situ hybridization showed that, in the absence of fully organized nucleoli, the three RNAs are diffusely distributed in the nucleus and partly associated with a number of small structures. Nucleolin and fibrillarin are also present in the anucleolate embryos as in normal embryos, although there is less nucleolin mRNA in the former. The two nucleolar proteins were localized by immunofluorescence microscopy. Fibrillarin, similar to its associated U3 and U15 snoRNAs, is diffusely distributed in the anucleolate nucleus and is partly associated with small structures, probably prenucleolar bodies and pseudonucleoli. Nucleolin also appears diffusely distributed in the nucleus with some spots of higher concentration, but with a different pattern with respect to fibrillarin.

A Xenopus laevis homologue of the La autoantigen binds the pyrimidine tract of the 5' UTR of ribosomal protein mRNAs in vitro: implication of a protein factor in complex formation.

In Xenopus and other vertebrates, ribosomal protein mRNAs share a common sequence in the 5' untranslated region (5' UTR), in particular a pyrimidine tract at the 5' end, which has been demonstrated to be involved in the translational regulation of this class of mRNAs. In previous studies, carried out in the Xenopus system, we demonstrated the specific binding of two proteins (57 kDa and 47 kDa) to the pyrimidine tract of the mRNAs for three different ribosomal proteins. Here, we show that the two binding proteins are in fact one; one being the cleavage product of the other. By immunoprecipitation and protein purification, this binding protein has been identified as the Xenopus homologue of the human La autoantigen, an RNA-binding protein previously reported to be implicated in RNA polymerase III transcription termination and in translation initiation of poliovirus and immunodeficiency virus type 1 RNAs. We show that the specific interaction of La with the 5' pyrimidine tract of ribosomal protein mRNA is mediated by a protease-sensitive factor, which, after assisting La-RNA binding, dissociates from the complex and becomes again available to promote further binding. We show that mutations in the 5' UTR pyrimidine tract, known to disrupt the translational control of ribosomal protein mRNA, severely impair La binding. Although a direct relationship between ribosomal protein mRNA translation and La binding is not yet available, the properties of the interaction suggest that La protein, possibly together with other components, might be involved in translational regulation.

Dynamic organization of splicing factors in adenovirus-infected cells.

Adenovirus infection affects the nuclear distribution of host splicing factors. Late phase-infected cells contain discrete clusters of small nuclear ribonucleoproteins (snRNPs) that are separate from centers containing the viral 72-kilodalton DNA-binding protein (72K protein). In the present study, we demonstrate that these snRNP clusters also contain splicing factors from the SR protein family. We show that a previously described monoclonal antibody, 3C5, detects SR proteins. Furthermore, we demonstrate that late region 3 transcription occurs at a maximal rate in infected cultures in which greater than 90% of the cells contain the snRNP clusters, indicating that such cells are actively transcribing their late genes. During the onset of the late phase, the intranuclear distribution of splicing factors is very different from that seen after the late phase is established. When late viral transcription commences, cells with snRNP clusters are less prevalent than in cultures that are maintaining maximum levels of late transcription. Instead, a cell type which shows snRNPs, concentrated in foci that also contain the viral 72K DNA-binding protein is detected. This cell type disappears from cultures by 18 to 20 h after a high-multiplicity infection. These results suggest a dynamic organization of splicing factors in infected cells that can be correlated to the status of viral gene expression. Our work also provides an explanation for the differing results that have been published concerning the organization of splicing factors in the adenovirus-infected cell nucleus (L. F. Jiménez-García and D. L. Spector, Cell 73:47-59, 1993). During the present study we observed that a monoclonal antibody against the SC-35 protein, which was used by Jiménez-García and Spector to study the localization of the SC-35 splicing factor in adenovirus-infected cells, cross-reacts with the adenovirus 72K DNA-binding protein and is thus unsuitable for this type of study.

Protein phosphatase 1 can modulate alternative 5' splice site selection in a HeLa splicing extract.

Recent studies using HeLa in vitro splicing extracts have shown that changes in the relative concentrations of constitutive protein splicing factors can affect the choice between competing 5' splice sites in alternatively spliced mammalian pre-mRNAs. Here we report that treatment of a HeLa splicing extract with human protein phosphatase 1 strongly inhibits formation of mRNA spliced to the distal 5' splice site while stimulating relative use of the proximal 5' splice site. This effect is not observed if spliceosomes assemble prior to protein phosphatase 1 treatment. These data show that alternative splicing in HeLa extracts can be mediated by changes in protein modification as well as by changes in the relative concentration of splicing factors. Changes in protein phosphorylation may thus provide a rapid mechanism for cells to respond to stimuli that require an alteration in alternative splicing patterns.

Interaction of proteins with the mRNA for ribosomal protein L1 in Xenopus: structural characterization of in vivo complexes and identification of proteins that bind in vitro to its 5'UTR.

Xenopus r-protein mRNAs are known to be coordinately regulated at the translational level. To find out if RNA/protein interactions are involved in this control mechanism, we have characterized the particles containing the translationally repressed rp-mRNA and we have investigated the proteins that specifically bind to this type of mRNA. By sedimentation analysis and isopycnic centrifugation we have found that the repressed rp-mRNAs are assembled in slow sedimenting complexes where the RNA is prevalent over the protein mass (2.3 to 1). This composition is maintained also after in vitro reconstitution of the particle. We carried out also a detailed analysis of in vitro RNA/protein complex formation by focusing our attention on the 5'UTR, very similar in different rp-mRNAs and important in the translational regulation. We describe specific interactions of L1 mRNA with four proteins. The binding site of two of them, 57 kD and 47 kD, is in the typical pyrimidine sequence at the 5' end and is position dependent. Proteins of the same size interact also with the analogous region of r-protein S1 and L14 mRNA, not with unrelated RNAs. Binding of two other proteins, 31 kD and 24 kD, in the downstream region of the 5'UTR was also observed. The most evident 57 kD protein has been partially purified. Although the binding of these proteins to the r-protein mRNA 5'UTR is specific, their involvement in the translation regulation remains to be proved.

Structure and expression of the nerve growth factor gene in Xenopus oocytes and embryos.

A large part of the coding portion of the Xenopus nerve growth factor (NGF) gene has been identified and cloned by the use of a chicken cDNA probe and its sequence has been determined. Comparison of the derived amino acid sequence of mature Xenopus NGF with that of other species showed a high conservation, whereas comparison of the prepropeptide showed large divergent regions alternated with short conserved regions. Expression of the NGF gene was examined during development of oocytes and embryos. Surprisingly, NGF mRNA was found in the oocyte; it is present in small previtellogenic as well as in fully grown oocytes. NGF mRNA, passed to the embryo at fertilization, is degraded before the gastrula stage and starts accumulating again around the stage of the neurula. The association of NGF mRNA with polysomes is indicative of NGF synthesis during oogenesis. In fact, by using antibodies against mouse NGF it was possible to reveal NGF molecules present as precursors. These molecules accumulate during oogenesis and are maintained in the embryos up to the blastula stage; a very faint band corresponding to a smaller size peptide is sometimes detected. A maternal role for the NGF can be proposed, although a possible activity of NGF in the oocyte cannot be ruled out.

Experimental changes in the amount of maternally stored ribosomes affect the translation efficiency of ribosomal protein mRNA in Xenopus embryo.

The amount of maternal free ribosomes in developing Xenopus embryos has been experimentally modified; an increase was obtained by microinjection of purified ribosomes into fertilized eggs, and a decrease was induced by treatment with a drug which reduces the amount of free ribosomes. The effect of this manipulation on the partition of the ribosomal protein mRNA (rp-mRNA) was analyzed during embryo development; it was observed that when ribosomes available for translation are in excess, polysome loading with rp-mRNA decreases. Conversely, when ribosomes are scarce, polysome loading of rp-mRNA increases. These experiments, which artificially stress events observed in the course of development, indicate that there is a relationship between the availability of ribosomes in the cells and the utilization of rp-mRNA for synthesis of ribosomal proteins, as already suggested by previous observations on r-protein synthesis during embryogenesis.

Expression of the gene for ribosomal protein L1 in Xenopus embryos: alteration of gene dosage by microinjection.

Cloned gene for Xenopus ribosomal protein L1 was injected into fertilized eggs, and its expression was analyzed during the period of embryo development when the mRNAs produced by the endogenous ribosomal protein genes are still silent due to a translational control. The injected genes replicated extensively, and a 10-fold excess of L1 mature transcript accumulated in the embryo. This was accompanied by a small amount of incompletely processed L1 RNA that still contained one out of nine introns, a molecule never observed in normal conditions. The excess mature L1 mRNA was distributed between polysomes and messenger ribonucleoproteins (mRNPs) in the same relative proportion observed in control embryos of the same stage. Therefore, more L1 mRNA was loaded onto polysomes and caused the appearance of L1 protein when this was not yet detectable in control embryos. The results suggest a relationship between the excess amount of L1 protein and the alteration in processing of its transcripts.

Ribosomal protein, histone and calmodulin mRNAs are differently regulated at the translational level during oogenesis of Xenopus laevis.

The localization of r-protein mRNA in subcellular compartments has been analysed. It was observed that the mRNA for a representative r-protein (L1) is diffuse in the cytoplasm, as shown by in situ hybridization experiments and that the distribution of rp-mRNA between polysomes and light mRNPs changes during oogenesis. In early oogenesis this mRNA is found mostly in subpolysomal fractions, whereas at the beginning of vitellogenesis (stage II) it becomes associated with polysomes where it remains in a constant amount at later stages. Histone and calmodulin mRNA, on the contrary, are mostly associated with non-polysomal fast-sedimenting particles throughout oogenesis. This suggests that the partition of different classes of mRNA between polysomes, light mRNP and heavy particles depends on their nature and might be determined by different requirements for these mRNAs during oogenesis.