Nested introns in an intron: Evidence of multi-step splicing in a large intron of the human dystrophin pre-mRNA

AbstractThe mechanisms by which huge human introns are spliced out precisely are poorly understood. We analyzed large intron 7 (110199 nucleotides) generated from the human dystrophin (DMD) pre-mRNA by RT-PCR. We identified branching between the authentic 5′ splice site and the branch point; however, the sequences far from the branch site were not detectable. This RT-PCR product was resistant to exoribonuclease (RNase R) digestion, suggesting that the detected lariat intron has a closed loop structure but contains gaps in its sequence. Transient and concomitant generation of at least two branched fragments from nested introns within large intron 7 suggests internal nested splicing events before the ultimate splicing at the authentic 5′ and 3′ splice sites. Nested splicing events, which bring the authentic 5′ and 3′ splice sites into close proximity, could be one of the splicing mechanisms for the extremely large introns

Alternative splicing produces structural and functional changes in CUGBP2

<p>Abstract</p> <p>Background</p> <p>CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons.</p> <p>Results</p> <p>The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (<it>R3δ</it>) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 <it>R3δ </it>isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the <it>ACTN1 </it>gene, these isoforms showed an opposite effect on the skipping of exon 11 in the <it>insulin receptor </it>gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.</p> <p>Conclusion</p> <p>Our results suggest that CUGBP2 regulates the splicing of <it>ACTN1 </it>and <it>insulin receptor </it>by different mechanisms. Alternative splicing of <it>CUGBP2 </it>exon 14 contributes to the regulation of the splicing of the <it>insulin receptor</it>. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.</p

‘Protected DNA Probes’ capable of strong hybridization without removal of base protecting groups

We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac4C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac6az8c7A). It was found that PDP containing ac4C and ac6az8c7A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness

Probe-on-carriers for oligonucleotide microarrays (DNA chips)

Oigonucleotide microarrays (DNA chips) are very efficient tools to analyze genotypes of patients or change in gene expressions between two different samples. However, there is no cost effective procedure to manufacture DNA chips. We are developing “probe-on-carriers”, immobilized oligonucleotide probes on solid phase to make DNA chips. In this procedure, each oligonucleotide is synthesized on a controlled porous glass carrier as a solid phase, and can be used as a probe for each sequence. This can be substantiated by technology for strictly controlled pore-size of porous glass. In fact, we found the sequence specific hybridization of probe-on-carrier with using porous glass of larger than 50nm pore diameter. The probe-on-carriers for wildtype and mutant p53 genes were hybridized with their complementary probes, respectively, but not with another probes. This result clearly demonstrated that the probe-on-carriers could recognize one-nucleotide substitutions of a gene. We found that the fixed probe-on-carriers on a slide glass still showed sequence specific hybridization. Therefore, we conclude that the probe-on-carriers are epoch-making materials for making DNA chip economically

C-to-U RNA Editing: A Site Directed RNA Editing Tool for Restoration of Genetic Code

The restoration of genetic code by editing mutated genes is a potential method for the treatment of genetic diseases/disorders. Genetic disorders are caused by the point mutations of thymine (T) to cytidine (C) or guanosine (G) to adenine (A), for which gene editing (editing of mutated genes) is a promising therapeutic technique. In C-to-Uridine (U) RNA editing, it converts the base C-to-U in RNA molecules and leads to nonsynonymous changes when occurring in coding regions; however, for G-to-A mutations, A-to-I editing occurs. Editing of C-to-U is not as physiologically common as that of A-to-I editing. Although hundreds to thousands of coding sites have been found to be C-to-U edited or editable in humans, the biological significance of this phenomenon remains elusive. In this review, we have tried to provide detailed information on physiological and artificial approaches for C-to-U RNA editing

NSSRs/TASRs/SRp38s function as splicing modulators via binding to pre-mRNAs

The genes for neural-salient serine/arginine-rich (NSSR) proteins 1 and 2 have been cloned from a neuronal differentiated embryocarcinoma cell line, P19. NSSRs contain an RNA recognition motif (RRM) at the N-terminal and several SR rich regions at the C-terminal resembling RS domains. We found that NSSRs associated with U1-70k, and determined the exon inclusion activity of NSSRs' C-terminals. First, the RRM was changed to the MS2 coat protein (MS2CP) and then, MS2 RNA stem-loops were inserted in the middle of the exon N of the clathrin light chain B minigene as an artificial exonic splicing enhancer to be recognized by the MS2CP. The modified exon N of the pre-mRNA was included by the MS2CP switched NSSR 1, but it was excluded by the MS2CP switched NSSR 2. Deletion analysis of the MS2CP switched NSSR1 suggested that the middle SR rich region was responsible for the activity of the modified exon N inclusion. Furthermore, the RRM domain of NSSRs recognized mRNAs. NSSRs were expressed in the nervous system, especially in cerebellar and hippocampal primordia, neopallial cortex, ventricular zone, retina, and olfactory epithelium and bulb at E15.5. Taken together, our results showed that NSSRs modulate alternative splicing via binding to pre-mRNAs during neural differentiation

Prediction of tertiary structure of NSSRs' RNA recognition motif and the RNA binding activity

RNAs possess potentials to become excellent bio-material because of their biochemical and biological activities. For instance, most RNA splicings are catalyzed by machinery including their own RNAs or other RNAs. The eukaryote machineries for splicing of pre-mRNA, which are called spliceosomes, are flexible and accurate for separating substrate and catalytic RNAs. Although RNAs themselves catalyze the splicing, spliceosomes are supported by many proteins. Furthermore, a great accuracy is required for the alternative splicing because there are choices available, which must be regulated in tissue-specifically and developmentally manners. Neural-salient serine/arginine-rich (NSSR) proteins 1 and 2 are candidates for supporting the accuracy of the splicing. The features of their amino acid sequences suggest that NSSRs are SR proteins, which bind to pre-mRNA and determine the splicing site. Since SR proteins have a RNA recognition motif or motives (RRM or RRMs), which binds to RNA, we predicted the secondary and tertiary structures of NSSRs' RRM by comparing them to RRMs of other proteins. The predicted structure suggested that the RNA binding activity of NSSRs' RRM is similar to the poly A binding protein (PABP). Moreover, to detect the targets for NSSR, mRNAs were obtained by screening them from murine brains with bacterial recombinant NSSRs' RRM and microarray experiments were conducted using these mRNAs. The results suggested that NSSRs bind specifically to particular pre-mRNAs and regulate the alternative splicing of the binding pre-mRNA

Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System

Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA

Effective RNA Knockdown Using CRISPR-Cas13a and Molecular Targeting of the EML4-ALK Transcript in H3122 Lung Cancer Cells

RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA&ndash;guide RNA complex residing at the 5&prime; of the crRNA. The activity of Cas13a was maximal for guide RNAs 24&ndash;30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70&ndash;76%), mCherry fluorescence (72%), and EML4-ALK at the protein (&gt;80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine