5' exon replacement and repair by spliceosome-mediated RNA trans-splicing

Spliceosome-mediated RNA trans-splicing (SMaRT) has been used previously to reprogram mutant endogenous CFTR and factor VIII mRNAs in human epithelial cell and tissue models and knockout mice, respectively. Those studies used 3' exon replacement (3'ER); a process in which the distal portion of RNA is reprogrammed. Here, we also show that the 5' end of mRNA can be completely rewritten by 5'ER. For proof-of-concept, and to test whether 5'ER could generate functional CFTR, we generated a mutant minigene target containing CFTR exons 10-24 (deltaF508) and a mini-intron 10, and a pretrans-splicing molecule (targeted to intron 10) containing CFTR exons 1-10 (+F508), and tested these two constructs in 293T cells for anion efflux transport. Cells cotransfected with target and PTM showed a consistent increase in anion efflux, but there was no response in control cells that received PTM or target alone. Using a LacZ reporter system to accurately quantify trans-splicing efficiency, we tested several unique PTM designs. These studies provided two important findings as follows: (1) efficient trans-splicing can be achieved by binding the PTM to different locations in the target, and (2) relatively few changes in PTM design can have a profound impact on trans-splicing activity. Tethering the PTM close to the target 3' splice site (as opposed to the donor site) and inserting an intron in the PTM coding resulted in a 65-fold enhancement of LacZ activity. These studies demonstrate that (1) SMaRT can be used to reprogram the 5' end of mRNA, and (2) efficiency can be improved substantially.     

Split introns in the genome of Giardia intestinalis are excised by spliceosome-mediated trans-splicing

Spliceosomal introns are hallmarks of most eukaryotic genomes and are excised from premature mRNAs by a spliceosome that is among the largest, and most complex, molecular machine in cells. The divergent unicellular eukaryote Giardia intestinalis, the causative agent of giardiasis, also possesses spliceosomes, but only four canonical (cis-spliced) introns have been identified in its genome to date. We demonstrate that this organism has a novel form of spliceosome-mediated trans-splicing of split introns that is essential for generating mature mRNAs for at least two important genes: one encoding a heat shock protein 90 (HSP90), which controls the conformation of a suite of cellular proteins, and the other encoding a dynein molecular motor protein, involved in the motility of eukaryotic flagella. These split introns have properties that distinguish them from other trans-splicing systems known within eukaryotes, suggesting that Giardia independently evolved a unique system to splice split introns.     

A novel spliceosome-mediated trans-splicing can change our view on genome complexity of the divergent eukaryote Giardia intestinalis

Although spliceosomal introns are an abundant landmark in eukaryotic genomes, the nuclear genome of the divergent eukaryote Giardia intestinalis, the causative agent of giardiasis, has been considered as “intron-poor” with only five canonical (cis-spliced) introns. However, three research groups (including ours) have independently reported a novel class of spliceosomal introns in the G. intestinalis genome. Three protein-coding genes are split into pieces in the G. intestinalis genome, and each of the partial coding regions was independently transcribed into polyadenylated premature mRNAs (pre-mRNAs). The two pre-mRNAs directly interact with each other by an intermolecular-stem structure formed between their non-coding portions, and are then processed into mature mRNAs by spliceosome-mediated trans-splicing. Here, we summarize the recently published works on split introns (“splintrons”) in the G. intestinalis genome, and then provide our speculation on the functional property of the Giardia spliceosomes based on the putative ratio of splintrons to canonical introns. Finally, we discuss a scenario for the transition from typical GT-AG boundaries to non-typical AT-AC boundaries in a particular splintron of Giardia.     

Genome-editing technologies for gene correction of hemophilia

Hemophilia is caused by various mutations in blood coagulation factor genes, including factor VIII (FVIII) and factor IX (FIX), that encode key proteins in the blood clotting pathway. Although the addition of therapeutic genes or infusion of clotting factors may be used to remedy hemophilia’s symptoms, no permanent cure for the disease exists. Moreover, patients often develop neutralizing antibodies or experience adverse effects that limit the therapy’s benefits. However, targeted gene therapy involving the precise correction of these mutated genes at the genome level using programmable nucleases is a promising strategy. These nucleases can induce double-strand breaks (DSBs) on genomes, and repairs of such induced DSBs by the two cellular repair systems enable a targeted gene correction. Going beyond cultured cell systems, we are now entering the age of direct gene correction in vivo using various delivery tools. Here, we describe the current status of in vivo and ex vivo genome-editing technology related to potential hemophilia gene correction and the prominent issues surrounding its application in patients with monogenic diseases.     

A multi-split mapping algorithm for circular RNA,splicing, trans-splicing and fusion detection

Numerous high-throughput sequencing studies have focused on detecting conventionally spliced mRNAs in RNA-seq data. However, non-standard RNAs arising through gene fusion, circularization or trans-splicing are often neglected. We introduce a novel, unbiased algorithm to detect splice junctions from single-end cDNA sequences. In contrast to other methods, our approach accommodates multi-junction structures. Our method compares favorably with competing tools for conventionally spliced mRNAs and, with a gain of up to 40% of recall, systematically outperforms them on reads with multiple splits, trans-splicing and circular products. The algorithm is integrated into our mapping tool segemehl (http://www.bioinf.uni-leipzig.de/Software/segemehl/).     

A computational investigation of kinetoplastid trans-splicing

Trans-splicing is an unusual process in which two separate RNA strands are spliced together to yield a mature mRNA. We present a novel computational approach which has an overall accuracy of 82% and can predict 92% of known trans-splicing sites. We have applied our method to chromosomes 1 and 3 of Leishmania major, with high-confidence predictions for 85% and 88% of annotated genes respectively. We suggest some extensions of our method to other systems.     

Naturally occurring heterologous trans-splicing of adenovirus RNA with host cellular transcripts during infection

The impact of viral infection on normal host RNA processing remains largely unexplored. We postulated that the high abundance of virally derived nuclear RNA in infected cells could impact host cell RNA splicing and viability. To test for aberrant RNA splicing we examined trans-splicing following infection with the replication-competent adenovirus mutant d11520 that lacks E1B 55 kDa protein. Trans-splicing was observed between viral RNA and several cellular precursor mRNAs, including beta-actin and glyceraldehyde-3-phosphate dehydrogenase. Using a tetracycline-inducible model system simulating viral trans-splicing activity we observed that overexpression of a trans-splicing RNA specifically inhibited cell proliferation. These results demonstrate that heterologous trans-splicing occurs naturally during adenovirus infection and suggest that trans-splicing may contribute to disruption of cell function.     

RNA editing in trans-splicing intron sequences of nad2 mRNAs in Oenothera mitochondria.

The complete open reading frame of subunit 2 of the NADH dehydrogenase in Oenothera mitochondria is split into five exons. The first two and the last three exons are encoded in distant genomic locations and are transcribed separately. Three tRNA genes coding for tRNA(Cys), tRNA(Asn), and tRNA(Tyr) are located upstream of the terminal three exons c, d, and e. The genomic distance, the interspersed tRNA genes, and the group II intron sequences flanking the two separated exons suggest trans-splicing to be required to connect exons b and c. Maturation of the mRNA includes RNA editing at 36 sites in the open reading frame. Three RNA editing events are observed in the split group II intron sequences. Two of these events allow after editing additional base pairings in the secondary structure, one in the stem of domain I, the other in the putative trans-pairing region of domain IV. These RNA editings may thus be involved in the trans-splicing reaction.     

SL1 trans-splicing specified by AU-rich synthetic RNA inserted at the 5' end of Caenorhabditis elegans pre-mRNA.

In Caenorhabditis elegans, pre-mRNAs of many genes are trans-spliced to one of two spliced leaders, SL1 or SL2. Some of those that receive exclusively SL1 have been characterized as having at their 5' ends outrons, AU-rich sequences similar to introns followed by conventional 3' splice sites. Comparison of outrons from many different SL1-specific C. elegans genes has not revealed the presence of any consensus sequence that might encode SL1-specificity. In order to determine what parameters influence the splicing of SL1, we performed in vivo experiments with synthetic splice sites. Synthetic AU-rich RNA, 51 nt or longer, placed upstream of a consensus 3' splice site resulted in efficient trans-splicing. With all sequences tested, this trans-splicing was specifically to SL1. Thus, no information beyond the presence of AU-rich RNA at least as long as the minimum-length C. elegans intron, followed by a 3' splice site, is required to specify trans-splicing or for strict SL1 specificity.