7SL RNA Research Articles

Analysis of spliceosomal complexes in Trypanosoma brucei and silencing of two splicing factors Prp31 and Prp43

In trypanosomatids all mRNAs undergo trans-splicing, whereas cis-splicing is restricted to a few transcripts. Trans-splicing is mechanistically similar to cis-splicing, however, little is known about the trans-splicing machinery and its underlying mechanism. In this study, we examined the involvement of splicing factors in cis- and trans-splicing by RNA interference (RNAi). Two factors (Prp31 and Prp43) were found to be essential for both pathways, suggesting that splicing factors are shared by these two reactions. We identified a 45S complex carrying pre-mRNA and all the U-snRNAs, including U1 and the SL RNA, suggesting that a single spliceosomal complex may potentially conduct both trans- and cis-splicing.

The Trypanosoma brucei signal recognition particle lacks the Alu-domain-binding proteins: purification and functional analysis of its binding proteins by RNAi

Trypanosomes are protozoan parasites that have a major impact on human health and that of livestock. These parasites represent a very early branch in the eukaryotic lineage, and possess unique RNA processing mechanisms. The trypanosome signal recognition particle (SRP) is also unusual in being the first signal recognition particle described in nature to be comprised of two RNA molecules, the 7SL RNA and a tRNA-like molecule. In this study, we further elucidated the unique properties of this particle. The genes encoding three SRP proteins (SRP19, SRP72 and SRP68) were identified by bioinformatics analysis. Silencing of these genes by RNAi suggests that the SRP-mediated protein translocation pathway is essential for growth. The depletion of SRP72 and SRP68 induced sudden death, most probably as a result of toxicity due to the accumulation of the pre-SRP in the nucleolus. Purification of the trypanosome particle to homogeneity, by TAP-tagging, identified four SRP proteins (SRP72, SRP68, SRP54 and SRP19), but no Alu-domain-binding protein homologs. This study highlights the unique features of the trypanosome SRP complex and further supports the hypothesis that the tRNA-like molecule present in this particle may replace the function of the Alu-domain-binding proteins present in many eukaryotic SRP complexes.

Expression of modified 7SL RNA gene in transgenic Solanum tuberosum plants

A modified plant 7SL RNA gene from Arabidopsis thaliana designated AHIIA63M was introduced into potato plants via Agrobacterium-mediated transformation. No transgenic plants could be obtained using pGPTV-based binary vectors where AHIIA63M gene driven by polIII promoter was located close to the polII promoter of the selection gene. Special binary vectors with matrix attachment region (MAR) elements had to be used for transformation to insulate polII and polIII promoters within T-DNA. The level of AHIIA63M RNA in transgenic plants was lower than the levels of transcripts of transgenes driven by RNA polymerase II. The level of AHIIA63M transcript in transgenic potato plants was tissue specific. The highest expression was detected in roots and gynoecium and the lowest in tubers. Moreover, non-specific promoter activity within the MAR element was revealed. This activity contributed to AHIIA63M transcription. This is the first report of expression of a modified 7SL RNA gene in transgenic plants and promoter activity within the MAR element.

Down-regulation of 7SL RNA Expression and Impairment of Vesicular Protein Transport Pathways by Leishmania Infection of Macrophages

The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display reverse transcription-PCR and cDNA microarray analyses. The genes that are down-regulated in mouse (J774G8) or human (U937) macrophages upon exposure to Leishmania include small RNA transcripts from the short interspersed element sequences. Among the short interspersed element RNAs that are down-regulated is 7SL RNA, which is the RNA component of the signal recognition particle. Because the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down-regulation of 7SL RNA may be significant in the establishment of infection by Leishmania in macrophage phagolysosomes. To evaluate whether down-regulation of 7SL RNA results in inhibition of signal recognition particle-mediated vesicular protein transport processes, we have tested and found that the targeting of proteins to the endoplasmic reticulum and plasma membrane and the secretion of proteins by macrophages are compromised in Leishmania-infected J774G8 and U937 cells. Knocking down 7SL RNA using small interfering RNA mimicked the effect of exposure of macrophages to Leishmania. The overexpression of 7SL RNA in J774G8 or U937 cells made these cells resistant to Leishmania infection, suggesting the possible biological significance of down-regulation of 7SL RNA synthesis in the establishment of infection by Leishmania. We conclude that Leishmania down-regulates 7SL RNA in macrophages to manipulate the targeting of many proteins that use the vesicular transport pathway and thus favors its successful establishment of infection in macrophages.

Plant 7SL RNA genes belong to type 4 of RNA polymerase III- dependent genes that are composed of mixed promoters

The genes transcribed by RNA polymerase III (pol III) display a great diversity in terms of promoter structure and are placed in four groups accordingly. Type 3 subset of pol III genes has promoter elements which reside entirely upstream of the coding region of the gene whereas type 4 consists of genes with mixed promoters that enclose intra- and extragenic regulatory sequences. Plant 7SL RNA genes have been previously classified as type 3 of pol III genes requiring an upstream sequence element and a canonical TATA box for transcriptional activity in transfected plant protoplasts. We have identified two novel functional control regions within the coding region of an Arabidopsis 7SL RNA gene (At7SL-1) that resemble tRNA gene-specific A and B boxes with respect to sequence and position. Single and multiple nucleotide substitutions in either of these regions resulted in a pronounced reduction of transcription activity in tobacco nuclear extract that was not caused by a decreased stability as shown by decay kinetics of wild type and mutant RNA transcripts. These findings suggest that plant 7SL RNA genes should be actually placed in type 4 of pol III-transcribed genes. As a consequence of substantially different upstream promoters utilized by plant and human pol III, in vitro transcription of 7SL RNA genes in heterologous systems is severely impaired. A chimeric human 7SL RNA gene that contains the 5' flanking region up to position -300 of At7SL-1 is yet transcribed with a reduced efficiency in tobacco extract when compared with the plant wild-type gene, supporting the notion that internal regulatory elements contribute to full activity.

Intragenomic spliced leader RNA array analysis of kinetoplastids reveals unexpected transcribed region diversity in Trypanosoma cruzi

The spliced leader RNA gene ( SL RNA) repeat is present in large multicopy arrays and has been used as a marker for the diversity of kinetoplastid protozoans. Intra-array variation could affect conclusions made using a randomly isolated repeat as a marker. We examined the Leishmania major (Friedlin) and Trypanosoma cruzi (CL Brener) genome projects for SL RNA repeat sequences in order to assess their homogeneity and the possible effects of sequence variation on taxonomic interpretation. Of the dozens of distinct sequence classes examined, no single copy would bias clustering analyses with regard to other closely related species or isolates. Six dimorphic sites within the T. cruzi transcribed region were found to be linked and are predicted to yield a heterogeneous SL RNA population. The variation that exists among the repeats paints a picture of the broad mechanisms of array maintenance and evolution where site-specific mutations in a single repeat may be spread throughout the array and recombined with existing repeats to create new sequence classes, all occurring under selective pressure to maintain or increase the fitness of the cell line in which these events occur.

Structural insights into SRP RNA: An induced fit mechanism for SRP assembly

Proper assembly of large protein-RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein-RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-A resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.

Grass Evolution Inferred from Chromosomal Rearrangements and Geometrical and Statistical Features in RNA Structure

The grasses (Poaceae) represent a monophyletic lineage that arose about 70 million years ago. The lineage contains about 10,000 species that differ widely in morphology and physiology. Species show striking differences in genome size, a feature important in the context of conservation of gene content and order (synteny and colinearity) and in the extension of genomic information directly from one grass species to another using comparative approaches. Grass diversification has been a contentious issue, as the exact branching order of the various subfamilies has been difficult to establish with standard methods. This motivated an evolutionary study of deep phylogenetic relationships based on the structure of coding and non-coding RNA molecules and on chromosomal rearrangements. Phylogenetic relationships in the grass family were inferred directly from the structure of RNA using cladistic principles and considerations in statistical mechanics. Coded attributes describing topological and thermodynamic information embedded in RNA molecules were treated as linearly ordered multi-state characters and were polarized by fixing the direction of character transformation toward molecular order. Intrinsically rooted phylogenies derived from the structure of signal recognition particle (SRP) RNA, the mRNA encoded by the early nodulation gene enod40, the small subunit of ribosomal RNA (rRNA), and the internal transcribed spacer ITS1 of rRNA established an order for the diversification of major grass lineages, suggesting a sister relationship of the Pooideae and the PACCAD clade. This same conclusion was reached when large-scale chromosomal rearrangements derived from the comparative genetic mapping of cereal genomes were studied. Chromosomal complements aligned in the most parsimonious manner allowed identification and coding of characters depicting chromosomal translocations, insertions, and linkage block arrangements and the reconstruction of phylogenetic trees based on large-scale chromosomal structure. Congruent reconstruction of deep branching relationships using geometrical and statistical features of RNA structure and orthology and large scale chromosomal recombination events support assumptions of polarization in character argumentation, and fail to falsify the claim that extant grass chromosomes can be considered combinations of linkage blocks of an ancestor of the rice genome. Congruence also suggests that the universal tendency toward order in RNA and the search for the most parsimonious organization of be genome architecture appear to be mutually supported drivers of molecular evolution. The study clarifies the relationship of major clades in the grasses, shows that phylogenetic history can be reconstructed effectively from the combinatorial exchange of chromosomal linkage blocks, and reveals considerable phylogenetic signal embedded in the structure of signal polypeptide-coding mRNA molecules, describing an instance where mRNA structure is the subject of strong evolutionary constraint.

Structural RNA has lower folding energy than random RNA of the same dinucleotide frequency

We present results of computer experiments that indicate that several RNAs for which the native state (minimum free energy secondary structure) is functionally important (type III hammerhead ribozymes, signal recognition particle RNAs, U2 small nucleolar spliceosomal RNAs, certain riboswitches, etc.) all have lower folding energy than random RNAs of the same length and dinucleotide frequency. Additionally, we find that whole mRNA as well as 5'-UTR, 3'-UTR, and cds regions of mRNA have folding energies comparable to that of random RNA, although there may be a statistically insignificant trace signal in 3'-UTR and cds regions. Various authors have used nucleotide (approximate) pattern matching and the computation of minimum free energy as filters to detect potential RNAs in ESTs and genomes. We introduce a new concept of the asymptotic Z-score and describe a fast, whole-genome scanning algorithm to compute asymptotic minimum free energy Z-scores of moving-window contents. Asymptotic Z-score computations offer another filter, to be used along with nucleotide pattern matching and minimum free energy computations, to detect potential functional RNAs in ESTs and genomic regions.

L1-mediated Retrotransposition of Murine B1 and B2 SINEs Recapitulated in Cultured Cells

SINEs are short interspersed nucleotide elements with transpositional activity, present at a high copy number (up to a million) in mammalian genomes. They are 80-400 bp long, non-coding sequences which derive either from the 7SL RNA (e.g. human Alus, murine B1s) or tRNA (e.g. murine B2s) polymerase III-driven genes. We have previously demonstrated that Alus very efficiently divert the enzymatic machinery of the autonomous L1 LINE (long interspersed nucleotide element) retrotransposons to transpose at a high rate. Here we show, using an ex vivo assay for transposition, that both B1 and B2 SINEs can be mobilized by murine LINEs, with the hallmarks of a bona fide retrotransposition process, including target site duplications of varying lengths and integrations into A-rich sequences. Despite different phylogenetic origins, transposition of the tRNA-derived B2 sequences is as efficient as that of the human Alus, whereas that of B1s is 20-100-fold lower despite a similar high copy number of these elements in the mouse genome. We provide evidence, via an appropriate nucleotide substitution within the B1 sequence in a domain essential for its intracellular targeting, that the current B1 SINEs are not optimal for transposition, a feature most probably selected for the host sake in the course of evolution.

Cofolga: a genetic algorithm for finding the common folding of two RNAs

In order to predict non-coding RNA genes and functions on the basis of genome sequences, accurate secondary structure prediction is useful. Although single-sequence folding programs such as mfold have been successful, it is of great importance to develop a novel approach for further improvement of the prediction performance. In the present paper, a secondary structure prediction method based on genetic algorithm, Cofolga, is proposed. The program developed performs folding and alignment of two homologous RNAs simultaneously. Cofolga was tested with a dataset composed of 13 tRNAs, seven 5S rRNAs, five RNase P RNAs, and five SRP RNAs; as a result, it turned out that the average prediction accuracies for the tRNAs, 5S rRNAs, RNase P RNAs, and SRP RNAs obtained by Cofolga with an optimal weight factor and default parameters were 83.6, 81.8, 73.5, and 67.7%, respectively. These results were superior to those obtained by a single-sequence folding based on free-energy minimization in which corresponding average prediction accuracies were 52.4, 47.4, 57.7, and 52.3%, respectively. Cofolga has a post-processing in which a single-sequence folding is performed after fixation of a predicted common structure; this post-processing enables Cofolga to predict a structure that is present in one of two RNAs alone. The executable files of Cofolga (for Windows/Unix/Mac) can be obtained by an e-mail request.

EVALUATION OF 7SL RNA GENE SEQUENCES FOR THE IDENTIFICATION OF LEISHMANIA SPP.

We evaluated the use of 7SL RNA gene sequences for the identification of Leishmania spp. A fragment (approximately 137 basepairs) of the 7SL RNA gene from 13 reference strains and 18 clinical isolates of 11 different Leishmania species was amplified and sequenced using conserved primers. Reference strains from each Leishmania spp. complex showed unique sequences. The nucleotide sequences were compared pairwise and a range of 81.0-99.3% intercomplex similarity was observed. Clinical isolates of the same species had sequences identical to the corresponding reference strains; thus, the intraspecies similarity was 100%. A phylogenetic tree derived from the 7SL RNA gene partial sequences was constructed and is in agreement with accepted phylogenetic schemes.

Spliced Leader RNA–Mediated trans-Splicing in Phylum Rotifera

In kinetoplastids, Euglena, and four metazoan phyla, trans-splicing has been described as a mechanism for the generation of mature messenger RNAs (mRNAs): 5'-ends of precursor mRNAs are replaced by a short spliced leader (SL) exon from a small SL RNA. Although the full phylogenetic range is unknown, trans-splicing has not been found in vertebrates, insects, plants, or yeast. In animal groups where it does occur, i.e., nematodes, cnidarians, platyhelminths, and primitive chordates, SL RNAs do not show sequence relatedness across phyla. The apparently sporadic phylogenetic distribution and the lack of SL RNA homology have led to opposing hypotheses on its evolution, involving either an ancient origin followed by loss in multiple lineages or independent acquisition in several taxa. Here we present evidence for the occurrence of trans-splicing in bdelloid rotifers (Bdelloidea, Rotifera). A common 23-nt sequence, representing the SL exon-diagnostic of SL RNA-mediated trans-splicing-was found at the 5'-end of at least 50%-65% of mRNAs from Adineta ricciae and Philodina sp. The trans-splicing pattern in bdelloid rotifers can be unusually complex, as observed in transcripts from a heat shock protein gene, hsp82-1, where the SL exon was spliced to three alternative positions. Bdelloid rotifer SL RNAs were found to be 105 or 106 nt long and comprised the SL sequence, a conserved splice donor site and an intron containing a putative spliceosome-binding motif. Intriguingly, some similarity of rotifer SL RNA sequence and predicted secondary structure was seen to that of the predominant SL1 RNA of nematodes, although it is unlikely that this demonstrates homology. In addition, sequence corresponding to the rotifer SL exon was found at the 5'-end of a number of full-length complementary DNA (cDNA) clones in a rice (Oryza sativa) database. None of these cDNAs gave a close match with homologous plant genes, suggesting that a small but significant portion of the rice expressed sequence tag database represents sequences derived from rotifers. In summary, the description of SL-mediated trans-splicing in Rotifera extends its representation to at least five metazoan phyla, making it increasingly probable that this is a phylogenetically widespread and therefore ancient phenomenon.

植物 7SL RNA 遺伝子の in vitro 転写解析

植物 7SL RNA 遺伝子の in vitro 転写解析

Signal recognition particle assembly in relation to the function of amplified nucleoli ofXenopusoocytes

The signal recognition particle (SRP) is a ribonucleoprotein machine that controls the translation and intracellular sorting of membrane and secreted proteins. The SRP contains a core RNA subunit with which six proteins are assembled. Recent work in both yeast and mammalian cells has identified the nucleolus as a possible initial site of SRP assembly. In the present study, SRP RNA and protein components were identified in the extrachromosomal, amplified nucleoli of Xenopus laevis oocytes. Fluorescent SRP RNA microinjected into the oocyte nucleus became specifically localized in the nucleoli, and endogenous SRP RNA was also detected in oocyte nucleoli by RNA in situ hybridization. An initial step in the assembly of SRP involves the binding of the SRP19 protein to SRP RNA. When green fluorescent protein (GFP)-tagged SRP19 protein was injected into the oocyte cytoplasm it was imported into the nucleus and became concentrated in the amplified nucleoli. After visiting the amplified nucleoli, GFP-tagged SRP19 protein was detected in the cytoplasm in a ribonucleoprotein complex, having a sedimentation coefficient characteristic of the SRP. These results suggest that the amplified nucleoli of Xenopus oocytes produce maternal stores not only of ribosomes, the classical product of nucleoli, but also of SRP, presumably as a global developmental strategy for stockpiling translational machinery for early embryogenesis.

A nomenclature for all signal recognition particle RNAs

The signal recognition particle (SRP) is a cytosolic ribonucleoprotein complex that guides secretory proteins to biological membranes in all organisms. The SRP RNA is at the center of the structure and function of the SRP. The comparison of the growing number of SRP RNA sequences provides a rich source for gaining valuable insight into the composition, assembly, and phylogeny of the SRP. In order to assist in the continuation of these studies, we propose an SRP RNA nomenclature applicable to the three divisions of life.

RNA silencing in plants by the expression of siRNA duplexes

In animal cells, stable RNA silencing can be achieved by vector-based small interfering RNA (siRNA) expression system, in which Pol III RNA gene promoters are used to drive the expression of short hairpin RNA, however, this has not been demonstrated in plants. Whether Pol III RNA gene promoter is capable of driving siRNA expression in plants is unknown. Here, we report that RNA silencing was achieved in plants through stable expression of short hairpin RNA, which was driven by Pol III RNA gene promoters. Using glucuronidase (GUS) transformed tobacco as a model system, the results demonstrated that 21 nt RNA duplexes, targeting at different sites of GUS gene, were stably expressed under the control of either human H1 or Arabidopsis 7SL RNA gene promoter, and GUS gene was silenced in 80% of siRNA transgenics. The severity of silencing was correlated with the abundance of siRNA expression but independent of the target sites and uridine residue structures in siRNA hairpin transcripts. Thus, the specific expression of siRNA provides a new system for the study of siRNA silencing pathways and functional genomics in plants. Moreover, the effectiveness of the human H1 promoter in a plant background suggested a conserved mechanism underlying Pol III complex functionality.

Identification of Chloroplast Signal Recognition Particle RNA Genes

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane in eukaryotes, the plasma membrane in bacteria and the thylakoid membrane in chloroplasts. In higher plants two different SRP-dependent mechanisms have been identified: one post-translational for proteins imported to the chloroplast and one co-translational for proteins encoded by the plastid genome. The post-translational chloroplast SRP (cpSRP) consists of the protein subunits cpSRP54 and cpSRP43. An RNA component has not been identified and does not seem to be required for the post-translational cpSRP. The co-translational mechanism is known to involve cpSRP54, but an RNA component has not yet been identified. Several chloroplast genomes have been sequenced recently, making a phylogenetically broad computational search for cpSRP RNA possible. We have analysed chloroplast genomes from 27 organisms. In higher plant chloroplasts, no SRP RNA genes were identified. However, eight plastids from red algae and Chlorophyta were found to contain an SRP RNA gene. These results suggest that SRP RNA forms a complex in these plastids with cpSRP54, reminiscent of the eubacterial SRP.

Identification of an RNA-binding Domain in Human SRP72

The signal recognition particle (SRP) is a ribonucleoprotein complex that plays a crucial role during the delivery of secretory proteins from the ribosome to the cell membrane. Among the six proteins of the eukaryotic SRP, the 72 kDa protein (SRP72) is the largest and least characterized. Polypeptides corresponding to various regions of the entire human SRP72 sequence were expressed in Escherichia coli, purified, and partially proteolyzed. Human SRP RNA bound with high affinity to a 63 amino acid residue region near the C terminus of SRP72. Mild treatment of the fragment with chymotrypsin abolished its RNA-binding activity. A conserved sequence with the consensus PDPXRWLPXXER was identified within a 56 amino acid residue RNA-binding domain. Sucrose gradient centrifugation and filter-binding analysis using mutant SRP RNAs showed that SRP72 bound to the moderately conserved portion of SRP RNA helix 5. Nine tetratricopeptide-like repeats (TPRs) poised to interact with other SRP or ribosomal proteins were predicted in the NH2-terminal region. These identifications assign two important functions to a large portion of SRP72 and demonstrate the RNA-binding capacity of the protein.

Novel upstream and intragenic control elements for the RNA polymerase III-dependent transcription of human 7SL RNA genes

In the human nuclear genome only a few copies coding for full-length 7SL RNA genes exist. The Hs7SL-1 gene has recently been classified as type 4 of RNA polymerase III (pol III)-transcribed genes as it was demonstrated that mutations in an external transcriptional activator (ATF) binding site and in an internal CG dinucleotide at positions +15/+16 reduced 7SL RNA expression in vivo and in vitro. We have extended the elucidation of external and internal promoter elements and have discovered two novel regulatory sequences: a TATA-like element in the upstream region and internal A and B box-like motifs. This study was greatly facilitated by the identification of a second, new functional human 7SL RNA gene which we called Hs7SL-3. Remarkably, Hs7SL-3 RNA is synthesized twice as efficiently as Hs7SL-1 in HeLa nuclear extract. Comparison of the upstream regions revealed the presence of two conserved elements in the two human 7SL RNA genes, an ATF/CRE binding site at –43 to –50 and a TATA-like box centered around position –25. Mutational analyses indicated that both external promoter elements are important for efficient transcription. In addition, two sequence motifs can be identified in Hs7SL-1 and Hs7SL-3 at positions 10–19 and 50–60, respectively, downstream of the transcription start site that resemble putative A and B boxes. Single and multiple nucleotide substitutions in these regions also influenced transcription activity to a great extent. The requirement of intragenic functional A and B boxes in combination with the external ATF/CRE and TATA-like promoter elements for the efficient transcription of human 7SL RNA genes is reminiscent of at least two other classes of pol III-transcribed genes in human cells, such as Epstein–Barr virus-encoded EBER and vault RNA genes.