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Alternative RNA splicing, a ubiquitous mechanism of gene regulation in eukaryotes, expands genome coding capacity and proteomic diversity. It has essential roles in all aspects of human physiology, including immunity. This review highlights the importance of RNA alternative splicing in regulating immune T cell function. We discuss how mutations that affect the alternative splicing of T cell factors can contribute to abnormal T cell function and ultimately lead to autoimmune diseases. We also explore the potential applications of strategies that target the alternative splicing changes of T cell factors. These strategies could help design therapeutic approaches to treat autoimmune disorders and improve immunotherapy.

In this commentary we briefly summarize early work on circular RNAs derived from spliceosome mediated circularization. We highlight how this early work inspired work on the basic mechanisms of nuclear RNA splicing, the possible function of circular RNAs and the potential uses of circular RNAs as tools in biomedicine. Recent developments in the study of circular RNAs, summarized in this volume, have brought these questions back to the foreground.

The regulated splicing of fibroblast growth factor receptor-2 (FGFR2) transcripts leads to tissue-specific expression of distinct receptor isoforms. These isoforms contain two different versions of the ligand binding Ig-like domain III, which are encoded by exon IIIb or exon IIIc. The mutually exclusive use of exon IIIb and exon IIIc can be recapitulated in tissue culture using DT3 and AT3 rat prostate carcinoma cells. We used this well-characterized system to evaluate the precision and accuracy of the RNA invasive cleavage assay to specifically measure FGFR2 alternative splicing outcomes. Experiments presented here demonstrated that the RNA invasive cleavage assay could specifically detect isoforms with discrimination levels that ranged from 1 in 5 x 10(3) to 1 in 10(5). Moreover the assay could detect close to 0.01 amole of FGFR2 RNAs. The assay detected the expected levels of transcripts containing either exon IIIb or IIIc, but, surprisingly, it detected high levels of IIIb-IIIc double inclusion transcripts. This finding, which has important implications for the role of exon silencing and of mRNA surveillance mechanisms, had been missed by RT-PCR. Additionally, we used the RNA invasive cleavage assay to demonstrate a novel function for the regulatory element IAS2 in repressing exon IIIc inclusion. We also show here that purification of RNA is not necessary for the invasive cleavage assay, because crude cell lysates could be used to accurately measure alternative transcripts. The data presented here indicate that the RNA invasive cleavage assay is an important addition to the repertoire of techniques available for the study of alternative splicing.

Protein-RNA interactions play indispensable structural, catalytic, and regulatory roles within the cell. Understanding their physical association in vivo provides valuable insight into their assembly, function, and regulation in the cellular milieu. Inspired by the chromatin immunoprecipitation assay, we have developed a ribonucleoprotein (RNP) immunoprecipitation assay to study RNA-protein interactions in vivo. This method takes advantage of the highly reactive, reversible crosslinker formaldehyde, combined with high-stringency immunoprecipitation to identify specific RNAs associated with a given protein. The RNP immunoprecipitation (RIP) assay was developed using RNA-protein interactions of hepatitis delta virus (HDV) as a model system. HDV is an RNA virus with a single-stranded circular RNA genome that encodes one viral protein, hepatitis delta antigen (HDAg). The high affinity of HDAg for the HDV RNA genome, combined with the well-characterized anti-HDAg antibodies, made this system a logical starting point for the development of the RIP assay. Cells with replicating HDV were crosslinked with formaldehyde and the HDV RNPs were immunoprecipitated using anti-HDAg antibodies. The crosslinks were then reversed by heat treatment, and the immunoprecipitated HDV RNAs were identified by reverse transcription polymerase chain reaction (RT-PCR). The specificity of this assay was tested using HDV mutants and heterologous antibodies for immunoprecipiation followed by RT-PCR with HDV-specific primers. This experiment showed no nonspecific immunoprecipitation of the HDV RNPs. The method was tested further using protein-RNA interactions known to exist in the U1 snRNP. The results indicate that the RIP assay is a powerful tool to identify RNA-protein interactions in vivo and has the potential to unravel the cellular network of RNP complexes in their native setting., ((c) 2002 Elsevier Science (USA).)

Flaviviruses are enveloped arthropod-borne viruses with a single-stranded, positive-sense RNA genome that can cause serious illness in humans and animals. The 11 kb 50 capped RNA genome consists of a single open reading frame (ORF), and is flanked by 50 and 30 untranslated regions (UTR). The ORF is a polyprotein that is processed into three structural and seven non-structural proteins. The UTRs have been shown to be important for viral replication and immune modulation. Both of these regions consist of elements that are essential for genome cyclization, resulting in initiation of RNA synthesis. Genome mutation studies have been employed to investigate each component of the essential elements to show the necessity of each component and its role in viral RNA replication and growth. Furthermore, the highly structured 30UTR is responsible for the generation of subgenomic flavivirus RNA (sfRNA) that helps the virus evade host immune response, thereby affecting viral pathogenesis. In addition, changes within the 30UTR have been shown to affect transmissibility between vector and host, which can influence the development of vaccines. [ABSTRACT FROM AUTHOR]

Flaviviruses are a genus of (+)ssRNA (positive ssRNA) enveloped viruses that replicate in the cytoplasm of cells of diverse species from arthropods to mammals. Many are important human pathogens such as DENV-1-4 (dengue virus types 1- 4), WNV (West Nile virus), YFV (yellow fever virus), JEV (Japanese encephalitis virus) and TBEV (tick-borne encephalitis). Given their RNA genomes it is not surprising that flaviviral life cycles revolve around critical RNA transactions. It is these we highlight in the present article. First, we summarize the mechanisms governing flaviviral replication and the central role of conserved RNA elements and viral protein-RNA interactions in RNA synthesis, translation and packaging. Secondly, we focus on how host RNA-binding proteins both benefit and inhibit flaviviral replication at different stages of their life cycle in mammalian hosts. Thirdly, we cover recent studies on viral non-coding RNAs produced in flavivirus-infected cells and how these RNAs affect various aspects of cellular RNA metabolism. Together, the article puts into perspective the central role of flaviviral RNAs in modulating both viral and cellular functions. [ABSTRACT FROM AUTHOR]

Background: HIV-1 relies on several host proteins for productive viral transcription. HIV-1 Tat-specific factor 1 (Tat-SF1) is among these cofactors that were identified by in vitro reconstituted transcription reactions with immunodepleted nuclear extracts. At the onset of this work, the prevailing hypothesis was that Tat-SF1 was a required cofactor for the viral regulatory protein, Tat; however, this had not previously been formally tested in vivo. Methodology/Principal Findings: To directly address the involvement of Tat-SF1 in HIV-1 gene expression, we depleted Tat-SF1 in HeLa cells by conventional expression of shRNAs and in T- Rex -293 cells containing tetracycline-inducible shRNAs targeting Tat-SF1. We achieved efficient depletion of Tat-SF1 and demonstrated that this did not affect cell viability. HIV-1 infectivity decreased in Tat-SF1-depleted cells, but only when multiple rounds of infection occurred. Neither Tat-dependent nor basal transcription from the HIV-1 LTR was affected by Tat-SF1 depletion, suggesting that the decrease in infectivity was due to a deficiency at a later step in the viral lifecycle. Finally, Tat-SF1 depletion resulted in an increase in the ratio of unspliced to spliced viral transcripts. Conclusions/Significance: Tat-SF1 is not required for regulating HIV-1 transcription, but is required for maintaining the ratios of different classes of HIV-1 transcripts. These new findings highlight a novel, post-transcriptional role for Tat-SF1 in the HIV-1 life cycle. [ABSTRACT FROM AUTHOR]

Dengue fever is the most frequent arthropod-borne viral disease of humans, with almost half of the world’s population at risk of infection. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make dengue fever a global public health threat. Given their compact genomes, dengue viruses (DENV-1–4) and other flaviviruses probably require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors, have been identified. Here we identify insect host factors required for DENV-2 propagation, by carrying out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 double-stranded RNA library. This screen identified 116 candidate dengue virus host factors (DVHFs). Although some were previously associated with flaviviruses (for example, V-ATPases and α-glucosidases), most of the DVHFs were newly implicated in dengue virus propagation. The dipteran DVHFs had 82 readily recognizable human homologues and, using a targeted short-interfering-RNA screen, we showed that 42 of these are human DVHFs. This indicates notable conservation of required factors between dipteran and human hosts. This work suggests new approaches to control infection in the insect vector and the mammalian host. [ABSTRACT FROM AUTHOR]

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3' end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9-exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10- tau RNA into exon 10+ tau RNA could be achieved with ≈34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing. [ABSTRACT FROM AUTHOR]

Examines how spliceosomes can recombine primary transcripts in trans to reprogram messenger RNA (mRNA). Description of the reprogramming of mRNA; Ways to accomplish mRNA reprogramming; Description of spliceosomes; Components in the spliceosome-mediated trans-splicing reactions.

In human cells, the majority of the primary transcripts synthesized by RNA polymerase II contain interruptions in the coding sequences, which are known as introns. The spliceosome, a macromolecular enzyme, precisely and efficiently removes these introns in a process known as pre-messenger RNA splicing. This enzyme is also capable of recombining two RNAs in trans in a process known as spliceosome-mediated RNA trans-splicing. In this review, we highlight technologies that take advantage of this RNA trans-splicing, from gene therapy to genomics. [ABSTRACT FROM AUTHOR]

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice.Trans-splicing between a cancer-associated pre-mRNA encoding the β-subunit of human chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significantβ-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy. [ABSTRACT FROM AUTHOR]

Report on an ancient, archaeal splicing endonuclease which provides a promising alternative for gene therapy. Encumbrance of gene therapy with everyday hurdles; Reliance on the evolution of novel approaches and techniques; Need of a third and significantly different method to revise messages; Possibility of recombining RNA molecules together to repair messenger RNAs showing a promising avenue for therapeutic exploration.

The global spread of dengue virus (DENV) infections has increased viral genetic diversity, some of which appears associated with greater epidemic potential. The mechanisms governing viral fitness in epidemiological settings, however, remain poorly defined. We identified a determinant of fitness in a foreign dominant (PR-2B) DENV serotype 2 (DENV-2) clade, which emerged during the 1994 epidemic in Puerto Rico and replaced an endemic (PR-1) DENV-2 clade. The PR-2B DENV-2 produced increased levels of subgenomic flavivirus RNA (sfRNA) relative to genomic RNA during replication. PR-2B sfRNA showed sequence- dependent binding to and prevention of tripartite motif 25 (TRIM25) deubiquitylation, which is critical for sustained and amplified retinoic acid-inducible gene 1 (RIG-I)-induced type I interferon expression. Our findings demonstrate a distinctive viral RNA-host protein interaction to evade the innate immune response for increased epidemiological fitness [ABSTRACT FROM AUTHOR]

An abstract of the article "Spliceosome Mediated RNA Trans-Splicing (SMaRTTM) Strategy to Increase the Production of Human Apolipoprotein (ApoA1)," by M. Puttaraju and colleagues is presented.