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The removal of introns from the primary transcripts of protein-coding genes is accomplished by the spliceosome, a large macromolecular complex of which small nuclear RNAs (snRNAs) are crucial components. Following the recent sequencing of the honeybee (Apis mellifera) genome, we used various computational methods, ranging from sequence similarity s...
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Context 1
... of the U11/U12 snRNP proteins first characterized in the human U11/U12 snRNP ( Will et al. 2004) and conserved broadly through eukaryotes ( Lorkovic et al. 2005) are absent from Drosoph- ila ( Schneider et al. 2004). We searched for genes encoding these U11/U12 proteins in the 11 insect genomes using translated BLAST searches (Table 2). All six Drosophila species seem to lack the 31K and 35K proteins, yet these same genes are present in the other five insect species, including the two mosquito genomes. ...
Context 2
... large gene family is known to harbor many U12 introns ( Wu and Krainer 1999), and our preliminary analysis suggests that the two introns present in the Apis gene are well conserved in different metazoan lineages (data not shown). The list of all the honeybee genes containing U12 introns is presented in Supplemental Table S2 at http://warta.bio.psu.edu/htt_doc/ Projects/snRNA/. ...
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Citations
... A small subset of these competition events involving adjacent U2-type and U12-type splice sites appear to have regulatory significance as suggested by Janice et al. (2013), who identified 18 twintron arrangements in the human genome that were evolutionarily conserved in vertebrates. An alternative hypothesis is that the nested introns may represent evolutionary intermediates in the process of minor to major intron conversion that has been suggested as an explanation for the low numbers of minor introns in present-day genomes (Burge et al., 1998;Mount et al., 2007;Lin et al., 2010;Janice et al., 2013;Moyer et al., 2020). ...
Many eukaryotic species contain two separate molecular machineries for removing non-coding intron sequences from pre-mRNA molecules. The majority of introns (more than 99.5% in humans) are recognized and excised by the major spliceosome, which utilizes relatively poorly conserved sequence elements at the 5′ and 3′ ends of the intron that are used for intron recognition and in subsequent catalysis. In contrast, the minor spliceosome targets a rare group of introns (approximately 0.5% in humans) with highly conserved sequences at the 5′ and 3′ ends of the intron. Minor introns coexist in the same genes with major introns and while the two intron types are spliced by separate spliceosomes, the two splicing machineries can interact with one another to shape mRNA processing events in genes containing minor introns. Here, we review known cooperative and competitive interactions between the two spliceosomes and discuss the mechanistic basis of the spliceosome crosstalk, its regulatory significance, and impact on spliceosome diseases.
... Persisting copies may then suffer divergence by mutations and sub-or neofunctionalization, or pseudogenization (Hughes and Nei 1992;Nei and Rooney 2005;Eirin-Lopez et al. 2012). Recent studies suggested a mixed effect of concerted and birth-and-death evolution to be involved in some multigene families dynamics (Mount et al. 2007;Freire et al. 2010;Pinhal et al. 2011;Merlo et al. 2012; Bardella and Cabral-de-Mello 2018;Zhang et al. 2021). ...
Multigene families are essential components of eukaryotic genomes and play key roles either structurally and functionally. Their modes of evolution remain elusive even in the era of genomics, because multiple multigene family sequences coexist in genomes, particularly in large repetitive genomes. Here, we investigate how the multigene families 18S rDNA, U2 snDNA, and H3 histone evolved in ten species of Schistocerca grasshoppers with very large and repeat-enriched genomes. Using sequenced genomes and FISH mapping, we find substantial differences for the multigene families, including the number of chromosomal clusters, changes in sequence abundance and nucleotide composition, pseudogenization, and association with transposable elements (TEs). The intra-genomic analysis of S. gregaria using long-read sequencing and genome assembly unveils conservation for H3 histone and recurrent pseudogenization for 18S rDNA and U2 snDNA, likely promoted by association with TEs and sequence truncation. Remarkably, TEs were frequently associated with truncated copies and were also among the most abundant in the genome, and revealed signatures of recent activity. Our findings suggest a combined effect of concerted and birth-and-death models driving the evolution of multigene families in Schistocerca over the last eight million years, and the occurrence of intra- and inter-chromosomal rearrangements shaping their chromosomal distribution. Despite the conserved karyotype in Schistocerca, our analysis highlights the extensive reorganization of repetitive DNAs in Schistocerca, contributing to the advance of comparative genomics for this important grasshopper genus.
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... RT-PCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA) using a miRcute Plus miRNA qPCR Kit (SYBR Green; Tiangen, Beijing, China) with specific primers (Supp Table 1 [online only]). U6 was chosen as an endogenous control for miRNA expression (Mount et al. 2007). Three technical replicates and three biological replicates were used to analyze the effects of dinotefuran treatment on miRNAs. ...
Honey bees are important pollinators of wild plants and crops. MicroRNAs (miRNAs) are endogenous regulators of gene expression. In this study, we initially determined that the lethal concentration 50 (LC50) of dinotefuran was 0.773 mg/l. Then, the expression profiles and differentially expressed miRNAs (DE miRNAs) in honey bee brains after 1, 5, and 10 d of treatment with the lethal concentration 10 (LC10) of dinotefuran were explored via deep small-RNA sequencing and bioinformatics. In total, 2, 23, and 27 DE miRNAs were identified after persistent exposure to the LC10 of dinotefuran for 1, 5, and 10 d, respectively. Some abundant miRNAs, such as ame-miR-375-3p, ame-miR-281-5p, ame-miR-3786-3p, ame-miR-10-5p, and ame-miR-6037-3p, were extremely significantly differentially expressed. Enrichment analysis suggested that the candidate target genes of the DE miRNAs are involved in the regulation of biological processes, cellular processes, and behaviors. These results expand our understanding of the regulatory roles of miRNAs in honey bee Apis mellifera (Hymenopptera: Apidae) responses to neonicotinoid insecticides and facilitate further studies on the functions of miRNAs in honey bees.
... Furthering the work of Mount et al. 15 and Konet et al., 6 we have demonstrated a pipeline for cell culture verification of Pol III promoter sequences in Culicine mosquitoes. In these experiments, we showed that Pol III promoter sequences from closely related species can be used to drive high levels of noncoding RNA expression in mosquito species of interest. ...
CRISPR-Cas9-based "gene drive" technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to date-and an expanded molecular toolbox with which to build them-will be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives.
... In 2004, Scamborova et al. showed that the nested major intron is preferentially spliced during early embryogenesis, while the outer minor intron is preferentially spliced in later embryonic development (Scamborova et al., 2004). Genes with similar twintron architectures have been identified in numerous insect and vertebrate genomes, indicating that this type of isoform regulation may be relatively common (Mount et al., 2007;Janice et al., 2013). Other MIGs have staggered majorand minor-type consensus sequences, such that the decision to recruit the major vs. the minor spliceosome would result in mutually exclusive exon selection (Letunic et al., 2002;Chang et al., 2007;Janice et al., 2013). ...
Minor introns constitute <0.5% of the introns in the human genome and have remained an enigma since their discovery. These introns are removed by a distinct splicing complex, the minor spliceosome. Both are ancient, tracing back to the last eukaryotic common ancestor (LECA), which is reflected by minor intron enrichment in specific gene families, such as the mitogen activated-protein kinase kinases, voltage-gated sodium and calcium ion channels, and E2F transcription factors. Most minor introns occur as single introns in genes with predominantly major introns. Due to this organization, minor intron-containing gene (MIG) expression requires the coordinated action of two spliceosomes, which increases the probability of missplicing. Thus, one would expect loss of minor introns via purifying selection. This has resulted in complete minor intron loss in at least nine eukaryotic lineages. However, minor introns are highly conserved in land plants and metazoans, where their importance is underscored by embryonic lethality when the minor spliceosome is inactivated. Conditional inactivation of the minor spliceosome has shown that rapidly dividing progenitor cells are highly sensitive to minor spliceosome loss. Indeed, we found that MIGs were significantly enriched in a screen for genes essential for survival in 341 cycling cell lines. Here, we propose that minor introns inserted randomly into genes in LECA or earlier and were subsequently conserved in genes crucial for cycling cell survival. We hypothesize that the essentiality of MIGs allowed minor introns to endure through the unicellularity of early eukaryotic evolution. Moreover, we identified 59 MIGs that emerged after LECA, and that many of these are essential for cycling cell survival, reinforcing our essentiality model for MIG conservation. This suggests that minor intron emergence is dynamic across eukaryotic evolution, and that minor introns should not be viewed as molecular fossils. We also posit that minor intron splicing was co-opted in multicellular evolution as a regulatory switch for en masse control of MIG expression and the biological processes they regulate. Specifically, this mode of regulation could control cell proliferation and thus body size, an idea supported by domestication syndrome, wherein MIGs are enriched in common candidate animal domestication genes.
... cDNA synthesis was performed using the miRcute Plus miRNA First-Strand cDNA synthesis kit (Tiangen) following the manufacturer's recommended protocol. RT-qPCR was performed using the miRcute Plus miRNA qPCR kit according to the manufacturer's instructions, and U6 small non-coding RNA was used as the reference gene in the RT-qPCR analysis [36]. The relative expression of the miRNAs was determined using the comparative quantity (2 −∆∆CT ) method [37]. ...
Bumblebees are important insect pollinators for many wildflowers and crops. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that regulate different biological functions in insects. In this study, the miRNAs in the heads of the three castes of the bumblebee Bombus lantschouensis were identified and characterized by small RNA deep sequencing. The significant differences in the expression of miRNAs and their target genes were analyzed. The results showed that the length of the small RNA reads from males, queens, and workers was distributed between 18 and 30 nt, with a peak at 22 nt. A total of 364 known and 89 novel miRNAs were identified from the heads of the three castes. The eight miRNAs with the highest expressed levels in males, queens, and workers were identical, although the order of these miRNAs based on expression differed. The male vs. queen, male vs. worker, and worker vs. queen comparisons identified nine, fourteen, and four miRNAs with significant differences in expression, respectively. The different castes were clustered based on the differentially expressed miRNAs (DE miRNAs), and the expression levels of the DE miRNAs obtained by RT-qPCR were consistent with the read counts obtained through Solexa sequencing. The putative target genes of these DE miRNAs were enriched in 29 Gene Ontology (GO) terms, and catalytic activity was the most enriched GO term, as demonstrated by its association with 2837 target genes in the male vs. queen comparison, 3535 target genes in the male vs. worker comparison, and 2185 target genes in the worker vs. queen comparison. This study highlights the characteristics of the miRNAs in the three B. lantschouensis castes and will aid further studies on the functions of miRNAs in bumblebees.
... 10 16 ) (see Additional file 1: Figure S6B). Stronger thermodynamic stability of some lincRNAs might also be explained by elements conferred by short non-coding RNAs such as microRNAs or snRNAs [55]. ...
Background
The ever increasing availability of genomes makes it possible to investigate and compare not only the genomic complements of genes and proteins, but also of RNAs. One class of RNAs, the long noncoding RNAs (lncRNAs) and, in particular, their subclass of long intergenic noncoding RNAs (lincRNAs) have recently gained much attention because of their roles in regulation of important biological processes such as immune response or cell differentiation and as possible evolutionary precursors for protein coding genes. lincRNAs seem to be poorly conserved at the sequence level but at least some lincRNAs have conserved structural elements and syntenic genomic positions. Previous studies showed that transposable elements are a main contribution to the evolution of lincRNAs in mammals. In contrast, plant lincRNA emergence and evolution has been linked with local duplication events. However, little is known about their evolutionary dynamics in general and in insect genomes in particular.
Results
Here we compared lincRNAs between seven insect genomes and investigated possible evolutionary changes and functional roles. We find very low sequence conservation between different species and that similarities within a species are mostly due to their association with transposable elements (TE) and simple repeats. Furthermore, we find that TEs are less frequent in lincRNA exons than in their introns, indicating that TEs may have been removed by selection. When we analysed the predicted thermodynamic stabilities of lincRNAs we found that they are more stable than their randomized controls which might indicate some selection pressure to maintain certain structural elements. We list several of the most stable lincRNAs which could serve as prime candidates for future functional studies. We also discuss the possibility of de novo protein coding genes emerging from lincRNAs. This is because lincRNAs with high GC content and potentially with longer open reading frames (ORF) are candidate loci where de novo gene emergence might occur.
Conclusion
The processes responsible for the emergence and diversification of lincRNAs in insects remain unclear. Both duplication and transposable elements may be important for the creation of new lincRNAs in insects.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-017-0985-0) contains supplementary material, which is available to authorized users.
... Reciprocal blastn searches were performed using parameters that are more tolerant of gaps and mismatches than default parameters (Camacho, et al. 2009;Mount, et al. 2007). The first blastn search queried the set of 1,771 D. pseudoobscura lncRNA transcripts against the full D. melanogaster transcriptome (FlyBase r6.02) (St Pierre, et al. 2014). ...
Thousands of long noncoding RNAs (lncRNAs) have been annotated in eukaryotic genomes, but comparative transcriptomic approaches
are necessary to understand their biological impact and evolution. To facilitate such comparative studies in Drosophila, we identified and characterized lncRNAs in a second Drosophilid – the evolutionary model D. pseudoobscura. Using RNA-Seq and computational filtering of protein-coding potential, we identified 1,589 intergenic lncRNA loci in D. pseudoobscura. We surveyed multiple sex-specific developmental stages and found, like in D. melanogaster, increasingly prolific lncRNA expression through male development and an overrepresentation of lncRNAs in the testes. Other
trends seen in D. melanogaster, like reduced pupal expression, were not observed. Nonrandom distributions of female-biased and non-testis-specific male-biased
lncRNAs between the X chromosome and autosomes are consistent with selection-based models of gene trafficking to optimize
genomic location of sex-biased genes. The numerous testis-specific lncRNAs, however, are randomly distributed between the
X and autosomes, and we cannot reject the hypothesis that many of these are likely to be spurious transcripts. Finally, using
annotated lncRNAs in both species, we identified 134 putative lncRNA homologs between D. pseudoobscura and D. melanogaster and find that many have conserved developmental expression dynamics, making them ideal candidates for future functional analyses.
... First, due to the lack of a genetically tractable system, early studies were unable to interrogate their biological relevance. Second, sequence analysis of snRNA paralogs across evolution suggests that all multi-copy snRNA genes have undergone concerted evolution, i.e., members of a given snRNA gene family are more similar within a species than between species (Pavelitz et al. 1995(Pavelitz et al. , 1999Mount et al. 2007;Marz et al. 2008). Because stable orthologous gene groups do not persist over evolutionary time (groups are usually only detectable within a genus), the possibility of significant functional divergence remains in question. ...
Pre-mRNA splicing is a critical step in eukaryotic gene expression that contributes to proteomic, cellular and developmental complexity. Small nuclear (sn)RNAs are core spliceosomal components, however, the extent to which differential expression of snRNA isoforms regulates splicing is completely unknown. This is partly due to difficulties in the accurate analysis of the spatial and temporal expression patterns of snRNAs. Here, we use high throughput RNA-sequencing (RNA-seq) data to profile expression of four major snRNAs throughout Drosophila development. This analysis shows that individual isoforms of each snRNA have distinct expression patterns in the embryo, larva and pharate adult stages. Expression of these isoforms is more heterogeneous during embryogenesis, and as development progresses, a single isoform from each snRNA subtype gradually dominates expression. Despite the lack of stable snRNA orthologous groups during evolution, this developmental switching of snRNA isoforms also occurs in distantly related vertebrate species, such as Xenopus, mouse and human. Our results indicate that expression of snRNA isoforms is regulated, and lay the foundation for functional studies of individual snRNA isoforms.
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... Interestingly, these are the base pairs that are the most stringently conserved among all the PSEAs of both the Pol II-and Pol III-transcribed snRNA genes of D. melanogaster. In fact, these are also the most conserved bases of all the other insect species' PSEAs that have been analyzed (20,33). It is further worth noting that the cross-linking patterns most similar between the U1 and U6 PSEAs are the cross-links between the DNA and the Rb repeat, as opposed to the other domains of Dm-SNAP190 ( Figure 7). ...
The small nuclear RNA (snRNA) activating protein complex (SNAPc) is essential for transcription of genes that encode the snRNAs.
Drosophila melanogaster SNAPc (DmSNAPc) consists of three subunits (DmSNAP190, DmSNAP50 and DmSNAP43) that form a stable complex that recognizes
an snRNA gene promoter element called the PSEA. Although all three subunits are required for sequence-specific DNA binding
activity, only DmSNAP190 possesses a canonical DNA binding domain consisting of 4.5 tandem Myb repeats homologous to the Myb
repeats in the DNA binding domain of the Myb oncoprotein. In this study, we use site-specific protein–DNA photo-cross-linking
technology followed by site-specific protein cleavage to map domains of DmSNAP190 that interact with specific phosphate positions
in the U6 PSEA. The results indicate that at least two DmSNAP190 Myb repeats contact the DNA in a significantly different
manner when DmSNAPc binds to a U6 PSEA versus a U1 PSEA, even though the two PSEA sequences differ at only 5 of 21 nucleotide
positions. The results are consistent with a model in which the specific DNA sequences of the U1 and U6 PSEAs differentially
alter the conformation of DmSNAPc, leading to the subsequent recruitment of different RNA polymerases to the U1 and U6 gene
promoters.
