Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3 Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.     

Comprehensive characterization of somatic variants associated with intronic polyadenylation in human cancers

Somatic single nucleotide variants (SNVs) in cancer genome affect gene expression through various mechanisms depending on their genomic location. While somatic SNVs near canonical splice sites have been reported to cause abnormal splicing of cancer-related genes, whether these SNVs can affect gene expression through other mechanisms remains an open question. Here, we analyzed RNA sequencing and exome data from 4,998 cancer patients covering ten cancer types and identified 152 somatic SNVs near splice sites that were associated with abnormal intronic polyadenylation (IPA). IPA-associated somatic variants favored the localization near the donor splice sites compared to the acceptor splice sites. A proportion of SNV-associated IPA events overlapped with premature cleavage and polyadenylation events triggered by U1 small nuclear ribonucleoproteins (snRNP) inhibition. GC content, intron length and polyadenylation signal were three genomic features that differentiated between SNV-associated IPA and intron retention. Notably, IPA-associated SNVs were enriched in tumor suppressor genes (TSGs), including the well-known TSGs such as PTEN and CDH1 with recurrent SNV-associated IPA events. Minigene assay confirmed that SNVs from PTEN, CDH1, VEGFA, GRHL2, CUL3 and WWC2 could lead to IPA. This work reveals that IPA acts as a novel mechanism explaining the functional consequence of somatic SNVs in human cancer.     

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.     

Accumulation of GC donor splice signals in mammals

Abstract

The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing.

Reviewers

This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section.     

Identification of a Human Immunodeficiency Virus Type 2 (HIV-2) Encapsidation Determinant and Transduction of Nondividing Human Cells by HIV-2-Based Lentivirus Vectors

Although previous lentivirus vector systems have used human immunodeficiency virus type 1 (HIV-1), HIV-2 is less pathogenic in humans and is amenable to pathogenicity testing in a primate model. In this study, an HIV-2 molecular clone that is infectious but apathogenic in macaques was used to first define cis-acting regions that can be deleted to prevent HIV-2 genomic encapsidation and replication without inhibiting viral gene expression. Lentivirus encapsidation determinants are complex and incompletely defined; for HIV-2, some deletions between the major 5′ splice donor and the gag open reading frame have been shown to minimally affect encapsidation and replication. We find that a larger deletion (61 to 75 nucleotides) abrogates encapsidation and replication but does not diminish mRNA expression. This deletion was incorporated into a replication-defective, envelope-pseudotyped, three-plasmid HIV-2 lentivirus vector system that supplies HIV-2 Gag/Pol and accessory proteins in trans from an HIV-2 packaging plasmid. The HIV-2 vectors efficiently transduced marker genes into human T and monocytoid cell lines and, in contrast to a murine leukemia virus-based vector, into growth-arrested HeLa cells and terminally differentiated human macrophages and NTN2 neurons. Vector DNA could be detected in HIV-2 vector-transduced nondividing CD34⁺ CD38⁻ human hematopoietic progenitor cells but not in those cells transduced with murine vectors. However, stable integration and expression of the reporter gene could not be detected in these hematopoietic progenitors, leaving open the question of the accessibility of these cells to stable lentivirus transduction.     

Functional characterization of two novel splicing mutations of glucokinase gene associated with maturity-onset diabetes of the young type 2 (MODY2)

Two novel mutations in the glucokinase gene (GCK) have been identified in patients with maturity-onset diabetes of the young type-2 (MODY2), i.e., a C-for-G substitution at position ?1 of the acceptor splice site of intron 7 (c. 864-1G>C) and a synonymous c.666C>G substitution (GTC>GTG, p.V222V) at exon 6. An analysis of the splicing products obtained upon the transfection of human embryonic HEK293 cells with GCK minigene constructs carrying these mutations showed that both substitutions impaired normal splicing. As a result of c.864-1G>C, the usage of the normal acceptor site was blocked, which activated cryptic acceptor splice sites within intron 7 and generated several aberrant RNAs containing fragments of intron 7. The synonymous substitution c.666C>G created a novel donor splice site in exon 6, which results in the formation of an abnormal GCK mRNA with a 16-nucleotide deletion in exon 6. In vitro experiments on minigene splicing confirmed the inactivating effect of these mutations on glucokinase gene expression.     

A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5’ splice site identity

Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5’ GU and ending with 3’ AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and “custody” of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5’ splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5’ splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5’ splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5’ splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.     

A splice variant of Arabidopsis mitogen-activated protein kinase and its regulatory function in the MKK6–MPK13 pathway

Alternative splicing (AS) generates multiple forms of proteins. A role for AS in the plant mitogen-activated protein kinase (MAPK) cascade has not been clarified. In this study, we analyzed expression of 20 Arabidopsis MAPK genes by RT-PCR and found five that generate splice variants. The MPK13 gene, with six exons and five introns, generates at least three splice variants, one in which complete splicing of five introns occurs (MPK13 Full), and ones in which the 4th and 5th introns are retained (MPK13 I4 and I5). Translation products of the splice variants MPK13 Full and I4, were found in Arabidopsis tissues by Western blot. Yeast two-hybrid analysis and protein kinase assays of recombinant proteins showed that neither I4 nor I5 interacted with upstream MAPKKs, and neither had protein kinase activity. However, MPK13 I4 protein enhanced the MKK6-dependent activation of MPK13 Full, indicating the possibility of an additional mechanism to regulate the MAPK cascade by AS.     

Redirecting splicing with bifunctional oligonucleotides

Ectopic modulators of alternative splicing are important tools to study the function of splice variants and for correcting mis-splicing events that cause human diseases. Such modulators can be bifunctional oligonucleotides made of an antisense portion that determines target specificity, and a non-hybridizing tail that recruits proteins or RNA/protein complexes that affect splice site selection (TOSS and TOES, respectively, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use of TOSS and TOES has been restricted to a handful of targets. To generalize the applicability and demonstrate the robustness of TOSS, we have tested this approach on more than 50 alternative splicing events. Moreover, we have developed an algorithm that can design active TOSS with a success rate of 80%. To produce bifunctional oligonucleotides capable of stimulating splicing, we built on the observation that binding sites for TDP-43 can stimulate splicing and improve U1 snRNP binding when inserted downstream from 5′ splice sites. A TOES designed to recruit TDP-43 improved exon 7 inclusion in SMN2. Overall, our study shows that bifunctional oligonucleotides can redirect splicing on a variety of genes, justifying their inclusion in the molecular arsenal that aims to alter the production of splice variants.