A Model for Initiation of Mosaic HOX Gene Expression Patterns by Non-Coding RNAs in Early Embryos
There is growing appreciation for the role of non-coding (nc) RNA in regulation of HOX genes of Drosophila. Our data suggest that current models for activation by ncRNA at the bithorax complex (BX-C) genes are mistaken. We propose that bxd and iab ncRNAs repress coding HOX genes Ultrabithorax and abdominal-A, respectively, by transcriptional interference. It is not clear how regulation by non-coding RNAs is integrated with other regulatory mechanisms at HOX loci. We suggest that non-coding RNAs regulated by the trithorax group of epigenetic regulators have an early transient role in repression of HOX genes at the bithorax complex. Later, we propose that repression by HOX proteins, and members of the Polycomb group take over from repression by ncRNAs. We discuss emerging research questions in light of this model.
SMN1 and SMN2 represent two nearly identical copies of the Survival Motor Neuron gene in humans. Deletion of SMN1 coupled with the inability of SMN2 to compensate for the loss of SMN1 leads to Spinal Muscular Atrophy (SMA), a leading genetic cause of infant mortality. SMN2 holds the promise for cure of SMA if skipping of exon 7 during pre-mRNA splicing of SMN2 could be prevented. Previous reports have shown that a C to T mutation at the 6th position of exon 7 (C6U substitution in the transcript) is the primary cause of SMN2 exon 7 skipping. Cumulative evidence suggests that C6U abrogates an enhancer associated with SF2/ASF, as well as, creates a silencer associated with hnRNP A1. There is also evidence to suggest that C6U creates an extended inhibitory context (Exinct). Recently, an intronic hnRNP A1 motif, which is not conserved between two human SMN genes, have been implicated in skipping of SMN2 exon 7. However, mechanism by which two SMN2-specific hnRNP A1 motifs interact is not known. Systematic approaches including site-specific mutations, in vivo selections, RNA structure probing and antisense oligonucleotide microwalks have revealed additional cis-elements in exon 7 as well as in flanking intronic sequences. A unique intronic splicing silencer (ISS-N1) has emerged as an effective target for correction of SMN2 exon 7 splicing by short antisense oligonucleotides (ASOs). Low nanomolar concentrations of ASOs against ISS-N1 fully restored SMN2 exon 7 inclusion and increased levels of SMN in SMA patient cells. Such a robust antisense response could be due to accessibility of the target as well as the complete nullification of a strong inhibitory impact rendered by ISS-N1. Bifunctional oligonucelotides with capability to recruit stimulatory splicing factors in the vicinity of weak splice sites of exon 7 have also shown promise for correction of SMN2 exon 7 splicing. Considering an antisense-based strategy confers a unique advantage of sequence specificity, availability of many target worthy cis-elements holds strong potential for antisense-mediated therapy of SMA.
Isolation and Posttranscriptional Modification Analysis of Native BC1 RNA from Mouse Brain
We present a simple and general affinity method, based on size fractionation and nucleic acid complementarity, to isolate sufficient amounts of native RNA molecules for further physicochemical studies, such as modification state of nucleotides. In the case presented here, we purified four micrograms of dendritic neuronal BC1 RNA from 130 grams of mouse brain (initially yielding a total of 200 mg RNA). Directly combined liquid chromatography-electrospray ionization mass spectrometry (LC/MS) analysis revealed no base or sugar backbone modifications in native BC1 RNA, despite earlier indications that C-54 could be methylated in vitro (Cm5, position 54).
Localization of the Developmental Timing Regulator Lin28 to mRNP Complexes, P-bodies and Stress Granules
Lin28 is a conserved cytoplasmic protein with an unusual pairing of RNA-binding motifs: a cold shock domain and a pair of retroviral-type CCHC zinc fingers. In the nematode C. elegans, it is a regulator of developmental timing. In mammals, it is abundant in diverse types of undifferentiated cells. However, its molecular function is unknown. In pluripotent mammalian cells, Lin28 is observed in RNase-sensitive complexes with Poly(A)-Binding Protein, and in polysomal fractions of sucrose gradients, suggesting it is associated with translating mRNAs. Upon cellular stress, Lin28 locates to stress granules, which contain non-translating mRNA complexes. However, Lin28 also localizes to cytoplasmic Processing bodies, or P-bodies, sites of mRNA degradation and microRNA regulation, consistent with it acting to regulate mRNA translation or stability. Mutational analysis shows that Lin28s conserved RNA binding domains cooperate to put Lin28 in mRNPs, but that only the CCHC domain is required for localization to P-bodies. When both RNA-binding domains are mutated, Lin28 accumulates in the nucleus, suggesting that it normally shuttles from nucleus to cytoplasm bound to RNA. These studies are consistent with a model in which Lin28 binds mRNAs in the nucleus and accompanies them to ribosomes and P-bodies. We propose that Lin28 influences the translation or stability of specific mRNAs during differentiation.
Pre-mRNA 3 Cleavage is Reversibly Inhibited in Vitro by Cleavage Factor Dephosphorylation
During 3 end formation most pre-mRNAs undergo endonucleolytic cleavage and polyadenylation in the 3 untranslated region. Very little is known concerning the role that post-translational modifications play in the function and regulation of the factors required for 3 cleavage. Using the reconstituted pre-mRNA cleavage reaction, we find that non-specific dephosphorylation of HeLa cell nuclear extract leads to the loss of 3 cleavage activity. A variety of serine/threonine phosphatases inhibited cleavage activity, while a tyrosine phosphatase did not. When the three major cleavage factor activities CPSF, CstF and CFm (containing CFIm and CFIIm) were separated and dephosphorylated individually, only CFm was found to lose activity, indicating that the target of dephosphorylation resides within this fraction. In accordance with this result, only CFm was able to restore cleavage activity to HeLa nuclear extract whose 3 cleavage activity had been completely inactivated by dephosphorylation. We conclude that at least one subunit of either CFIm or CFIIm requires serine or threonine phosphorylation to function during 3 cleavage. Our data suggest that cleavage factor phosphorylation may serve as a regulatory on/off switch to control pre-mRNA 3 end formation.
Spinal Muscular Atrophy: Position and Functional Importance of the Branch Site Preceding SMN Exon 7
In spinal muscular atrophy, the SMN1 gene is deleted or destroyed by mutation, while the neigbouring, nearly identical SMN2 gene acts as a partial functional substitute. However, due to a single nucleotide exchange, the seventh exon of SMN2 is mostly excluded from the mature mRNA, and the resulting shorter protein is non-functional. Here, we map the previously uncharacterised intron 6 branch point by RT-PCR. Moreover we show that exon 7 inclusion can be either abolished or improved by mutations in this branch site region.
Mitochondrial tRNA Mutations: Clinical and Functional Perturbations
During the last decade, there has been a progressive accumulation of reports that connect the identification of specific mitochondrial tRNA gene mutations to severe disorders in human. As a result, mitochondrial tRNA genes and their products have emerged as novel and essential molecular markers for wide biochemical and genetic screenings among different human populations. So far, 139 pathogenic and 243 polymorphic mt tRNA mutations have been described and they have become the foreground of numerous case reports. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogeneous. They range from lesions of single tissues or structures to more severe impairements including myopathies, encephalomyopathies, cardiomyopathies, or complex multisystem syndromes. Moreover, the exact mechanisms by which biochemical cascades can be dramatically affected by mitochondrial tRNA mutations still remain uncharacterized. However and regardless of the vast amount of information that daily emerges, only few efforts have been carried out to systematically record all the mitochondrial tRNA-associated pathogenic mutations or polymorphisms. In this report, we summarize all the clinical phenotypes associated with mitochondrial tRNA pathogenic mutations that have been reported so far. In a next step we describe in detail all the pathogenic and polymorphic mutations that have been recorded so far and we categorize them per tRNA species and per associated disease. Finally, we discuss the impact of the frequency of mitochondrial tRNA mutations in general population surveys and we preview any relevant implications on the essential functional integrity of mitochondrial biochemical pathways.