The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila.

Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, we show that the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [35S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). We conclude that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export.     

Alternatively spliced p53 RNA in transformed and normal cells of different tissue types.

The alternatively spliced RNA species of tumor suppressor gene p53, containing an additional 96 bases derived from intron 10, is present at approximately 25 to 30% the level of regularly spliced p53 RNA in both normal epidermal and carcinoma cells. The presence of this alternatively spliced RNA in 10T1/2 fibroblast cells, mouse liver and testis suggests that this alternative splicing may be universal. The level of alternatively spliced p53 RNA was increased coordinately with that of regularly spliced p53 in 10T1/2 cells in response to epidermal growth factor. Immunoprecipitation analysis of epidermal cells using monoclonal antibodies which recognize different epitopes of p53 suggested that distinct p53 proteins may be translated from both RNA species. Considering previous observations on the potential importance of carboxyl terminal sequences in p53 function, knowledge of the ubiquitous presence of alternatively spliced p53 is important for future studies of p53 function in normal cells and in oncogenesis.     

cDNA structure, expression and nucleic acid-binding properties of three RNA-binding proteins in tobacco: occurrence of tissue-specific alternative splicing.

Three cDNAs encoding RNA-binding proteins were isolated from a tobacco (Nicotiana sylvestris) cDNA library. The predicted proteins (RGP-1) are homologous to each other and consist of a consensus-sequence type RNA-binding domain of 80 amino acids in the N-terminal half and a glycine-rich domain of 61-78 amino acids in the C-terminal half. Nucleic acid-binding assay using the in vitro synthesized RGP-1 protein confirmed that it is an RNA-binding protein. Based on its strong affinity for poly(G) and poly(U), the RGP-1 proteins are suggested to bind specifically to G and/or U rich sequences. All three genes are expressed in leaves, roots, flowers and cultured cells, however, the substantial amount of pre-mRNAs are accumulated especially in roots. Sequence analysis and ribonuclease protection assay indicated that significant amounts of alternatively spliced mRNAs, which are produced by differential selection of 5' splice sites, are also present in various tissues. Tissue-specific alternative splicing was found in two of the three genes. The alternatively spliced mRNAs are also detected in polysomal fractions and are suggested to produce truncated polypeptides. A possible role of this alternative splicing is discussed.     

Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity.     

A computational and experimental approach toward a priori identification of alternatively spliced exons

Alternative splicing is a powerful means of regulating gene expression and enhancing protein diversity. In fact, the majority of metazoan genes encode pre-mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Thus, an important part of determining the complete proteome of an organism is developing a catalog of all mRNA isoforms. Alternatively spliced exons are typically identified by aligning EST clusters to reference mRNAs or genomic DNA. However, this approach is not useful for genomes that lack robust EST coverage, and tools that enable accurate prediction of alternatively spliced exons would be extraordinarily useful. Here, we use comparative genomics to identify, and experimentally verify, potential alternative exons based solely on their high degree of conservation between Drosophila melanogaster and D. pseudoobscura. At least 40% of the exons that fit our prediction criteria are in fact alternatively spliced. Thus, comparative genomics can be used to accurately predict certain classes of alternative exons without relying on EST data.     

B-cell and plasma-cell splicing differences: a potential role in regulated immunoglobulin RNA processing

The immunoglobulin micro pre-mRNA is alternatively processed at its 3' end by competing splice and cleavage-polyadenylation reactions to generate mRNAs encoding the membrane-associated or secreted forms of the IgM protein, respectively. The relative use of the competing processing pathways varies during B-lymphocyte development, and it has been established previously that cleavage-polyadenylation activity is higher in plasma cells, which secrete IgM, than in B cells, which produce membrane-associated IgM. To determine whether RNA-splicing activity varies during B-lymphocyte development to contribute to micro RNA-processing regulation, we first demonstrate that micro pre-mRNA processing is sensitive to artificial changes in the splice environment by coexpressing SR proteins with the micro gene. To explore differences between the splice environments of B cells and plasma cells, we analyzed the splicing patterns from two different chimeric non-Ig genes that can be alternatively spliced but have no competing cleavage-polyadenylation reaction. The ratio of intact exon splicing to cryptic splice site use from one chimeric gene differs between several B-cell and several plasma-cell lines. Also, the amount of spliced RNA is higher in B-cell than plasma-cell lines from a set of genes whose splicing is dependent on a functional exonic splice enhancer. Thus, there is clear difference between the B-cell and plasma-cell splicing environments. We propose that both general cleavage-polyadenylation and general splice activities are modulated during B-lymphocyte development to ensure proper regulation of the alternative micro RNA processing pathways.     

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

Identification of novel snRNA-specific Sm proteins that bind selectively to U2 and U4 snRNAs in Trypanosoma brucei

In eukaryotes the seven Sm core proteins bind to U1, U2, U4, and U5 snRNAs. In Trypanosoma brucei, Sm proteins have been implicated in binding both spliced leader (SL) and U snRNAs. In this study, we examined the function of these Sm proteins using RNAi silencing and protein purification. RNAi silencing of each of the seven Sm genes resulted in accumulation of SL RNA as well as reduction of several U snRNAs. Interestingly, U2 was unaffected by the loss of SmB, and both U2 and U4 snRNAs were unaffected by the loss of SmD3, suggesting that these snRNAs are not bound by the heptameric Sm complex that binds to U1, U5, and SL RNA. RNAi silencing and protein purification showed that U2 and U4 snRNAs were bound by a unique set of Sm proteins that we termed SSm (specific spliceosomal Sm proteins). This is the first study that identifies specific core Sm proteins that bind only to a subset of spliceosomal snRNAs.     

Novel genomic cDNA hybrids produce effective RNA interference in adult Drosophila.

Drosophila melanogaster has been a premier genetic model system for nearly 100 years, yet lacks a simple method to disrupt gene expression. Here, we show genomic cDNA fusions predicted to form double-stranded RNA (dsRNA) following splicing, effectively silencing expression of target genes in adult transgenic animals. We targeted three Drosophila genes: lush, white, and dGq(alpha). In each case, target gene expression is dramatically reduced, and the white RNAi phenotype is indistinguishable from a deletion mutant. This technique efficiently targets genes expressed in neurons, a tissue refractory to RNAi in C. elegans. These results demonstrate a simple strategy to knock out gene function in specific cells in living adult Drosophila that can be applied to define the biological function of hundreds of orphan genes and open reading frames.     

Natural selection drives extremely rapid evolution in antiviral RNAi genes

RNA interference (RNAi) is perhaps best known as a laboratory tool. However, RNAi-related pathways represent an antiviral component of innate immunity in both plants and animals. Since viruses can protect themselves by suppressing RNAi, interaction between RNA viruses and host RNAi may represent an ancient coevolutionary "arms race." This could lead to strong directional selection on RNAi genes, but to date their evolution has not been studied. By comparing DNA sequences from different species of Drosophila, we show that the rate of amino acid evolution is substantially elevated in genes related to antiviral RNAi function (Dcr2, R2D2, and Ago2). They are among the fastest evolving 3% of all Drosophila genes; they evolve significantly faster than other components of innate immunity and faster than paralogous genes that mediate "housekeeping" functions. Based on DNA polymorphism data from three species of Drosophila, McDonald-Kreitman tests showed that this rapid evolution is due to strong positive selection. Furthermore, Dcr2 and Ago2 display reduced genetic diversity, indicative of a recent selective sweep in both genes. Together, these data show rapid adaptive evolution of the antiviral RNAi pathway in Drosophila. This is a signature of host-pathogen arms races and implies that the ancient battle between RNA viruses and host antiviral RNAi genes is active and significant in shaping RNAi function.