Halic M et al. (2009) Mol Cell "22G-RNAs in transposon silencing and centromere function."

Moazed D, Halic M

[Mol Cell, 2009]

Three recent papers (Gu et al., 2009; Claycomb et al., 2009; van Wolfswinkel et al., 2009) provide evidence that links a new class of small RNAs and Argonaute-associated complexes to centromere function and genome surveillance.

Bhattacharya S et al. (2013) J Agric Food Chem "Bioactive components from flowers of Sambucus nigra L. increase glucose uptake in primary porcine myotube cultures and reduce fat accumulation in Caenorhabditis elegans."

Oksbjerg N, Bhattacharya S, Christensen LP, Young JF, Christensen KB, Kristiansen K, Faergeman NJ, Grevsen K, Olsen LC

[J Agric Food Chem, 2013]

Obesity and insulin resistance in skeletal muscles are major features of type 2 diabetes. In the present study, we examined the potential of Sambucus nigra flower (elderflowers) extracts to stimulate glucose uptake (GU) in primary porcine myotubes and reduce fat accumulation (FAc) in Caenorhabditis elegans. Bioassay guided chromatographic fractionations of extracts and fractions resulted in the identification of naringenin and 5-O- caffeoylquinic acid exhibiting a significant increase in GU. In addition, phenolic compounds related to those found in elderflowers were also tested, and among these, kaempferol, ferulic acid, p-coumaric acid, and caffeic acid increased GU significantly. FAc was significantly reduced in C. elegans, when treated with elderflower extracts, their fractions and the metabolites naringenin, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, quercetin-3-O-5-acetylglycoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, and isorhamnetin-3-O-glucoside and the related phenolic compounds kaempferol and ferulic acid. The study indicates that elderflower extracts contain bioactive compounds capable of modulating glucose and lipid metabolism, suitable for nutraceutical and pharmaceutical applications.

Suzuki JM et al. (2022) PLoS Genet "A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5' splice site identity."

Cartwright-Acar CH, Osterhoudt K, Katzman S, Suzuki JM, Gomez DR, Zahler AM

[PLoS Genet, 2022]

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.

Fallmann J et al. (2016) Nucleic Acids Res "AREsite2: an enhanced database for the comprehensive investigation of AU/GU/U-rich elements."

Sedlyarov V, Hofacker IL, Kovarik P, Fallmann J, Tanzer A

[Nucleic Acids Res, 2016]

AREsite2 represents an update for AREsite, an on-line resource for the investigation of AU-rich elements (ARE) in human and mouse mRNA 3'UTR sequences. The new updated and enhanced version allows detailed investigation of AU, GU and U-rich elements (ARE, GRE, URE) in the transcriptome of Homo sapiens, Mus musculus, Danio rerio, Caenorhabditis elegans and Drosophila melanogaster. It contains information on genomic location, genic context, RNA secondary structure context and conservation of annotated motifs. Improvements include annotation of motifs not only in 3'UTRs but in the whole gene body including introns, additional genomes, and locally stable secondary structures from genome wide scans. Furthermore, we include data from CLIP-Seq experiments in order to highlight motifs with validated protein interaction. Additionally, we provide a REST interface for experienced users to interact with the database in a semi-automated manner. The database is publicly available at: http://rna.tbi.univie.ac.at/AREsite.

Rogers, Alicia Kathryn et al. MicroPubl Biol

Rogers, Alicia Kathryn, Phillips, Carolyn Marie

[MicroPubl Biol, 2020]

Mutator foci are perinuclear granules in the germline of Caenorhabditis elegans that are required for the amplification of 22G-small interfering RNAs (siRNAs) (Phillips et al., 2012). These mutator-dependent siRNAs act downstream of primary endogenous and exogenous siRNA pathways and are necessary for robust and heritable silencing (Pak et al., 2007; Sijen et al., 2007; Gu et al., 2009; Gent et al., 2010; Vasale et al., 2010; Phillips et al., 2012). There are numerous factors that have been identified that localize to Mutator foci and are required for mutator-dependent siRNA biogenesis. These mutator-class proteins include the core component of Mutator foci MUT-16, the nucleotidyl transferase MUT-2, the 3-5 exonuclease MUT-7, the DEAD-box RNA helicases MUT-14 and SMUT-1, the Zc3h12a-like ribonucleases RDE-8, NYN-1, and NYN-2, and two proteins of unknown function, MUT-15 and RDE-2 (Ketting et al., 1999; Tijsterman et al., 2002; Vastenhouw et al., 2003; Chen et al., 2005; Tops et al., 2005; Phillips et al., 2012; Phillips et al., 2014; Tsai et al., 2015). Additionally, the RNA-dependent RNA polymerase RRF-1 localizes to Mutator foci but is redundant with EGO-1 for mutator-dependent siRNA biogenesis (Phillips et al., 2012; Gu et al., 2009). It was previously shown that mutations in mutator-class genes are sterile at elevated temperature (Ketting et al., 1999; Zhang et al., 2011; Rogers and Phillips, 2020). Recently, we performed a brood size assay using wild-type and mut-16 hermaphrodites cultured at 20C. We found that compared to wild-type animals, mut-16 mutant animals lay fewer eggs (56% fewer eggs laid compared to wild-type animals), and of those eggs, fewer mut-16 mutant eggs hatch (81% of mut-16 mutant eggs hatch compared to wild-type, where 100% of the eggs hatch) (Rogers and Phillips, 2020). Furthermore, 100% of wild-type larvae successfully mature to adulthood, whereas only 85% of mut-16 mutant larvae mature to adulthood (Rogers and Phillips, 2020). The reduced hatching rates and larval arrest of mut-16 mutant animals had not been previously reported...

Wang CY et al. (2004) BMC Genomics "Molecular cloning and characterization of the mouse Acdp gene family."

Purohit S, Yang P, An H, Dong Z, Ling J, Gu JG, Wang CY, Guo D, Shi JD, She JX

[BMC Genomics, 2004]

BACKGROUND: We have recently cloned and characterized a novel gene family named ancient conserved domain protein (ACDP) in humans. To facilitate the functional study of this novel gene family, we have cloned and characterized Acdp, the mouse homologue of the human ACDP gene family. RESULTS: The four Acdp genes (Acdp1, Acdp2, Acdp3 and Acdp4) contain 3,631 bp, 3,244 bp, 2,684 bp and 2,743 bp of cDNA sequences, and encode deduced proteins of 951, 874, 713 and 771 amino acids, respectively. The mouse Acdp genes showed very strong homologies (>90%) in both nucleotide and amino acid sequences to their human counterparts. In addition, both nucleotide and amino acid sequences within the Ancient Conserved Domain (ACD) are highly conserved in many different taxonomic species. Particularly, Acdp proteins showed very strong AA homologies to the bacteria CorC protein (35% AA identity with 55% homology), which is involved in magnesium and cobalt efflux. The Acdp genes are widely expressed in all tissues tested except for Acdp1, which is only highly expressed in the brain with low levels of expression in kidney and testis. Immunostaining of Acdp1 in hippocampus neurons revealed a predominant localization on the plasma membrane. CONCLUSION: The Acdp genes are evolutionarily conserved in diverse species and ubiquitously expressed throughout development and adult tissues suggesting that Acdp may be an essential gene. Acdp showed strong homology to bacteria CorC protein and predominantly localized on the plasma membrane. These results suggest that Acdp is probably a family of proteins involved in ion transport in mammalian cells

Cevec M et al. (2008) Nucleic Acids Res "Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans."

Plavec J, Cevec M, Thibaudeau C

[Nucleic Acids Res, 2008]

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3'' untranslated region (3''-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem-loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA.

Rushforth AM et al. (1996) Mol Cell Biol "Splicing removes the Caenorhabditis elegans transposon Tc1 from most mutant pre-mRNAs."

Anderson P, Rushforth AM

[Mol Cell Biol, 1996]

The transposable element Tc1 is responsible for most spontaneous mutations that occur in many Caenorhabditis elegans strains. We analyzed the abundance and sequence of mRNAs expressed from five different Tc1 insertions within either hlh-1 (a MyoD homolog) or unc-54 (a myosin heavy chain gene). Each of the mutants expresses substantial quantities of mature mRNA in which most or all of Tc1 has been removed by splicing. Such mRNAs contain small insertions of Tc1 sequences and/or deletions of target gene sequences at the resulting spliced junctions. Most of these mutant mRNAs do not contain premature stop codons, and many are translated in frame to produce proteins that are functional in vivo. The number and variety of splice sites used to remove Tc1 from these mutant pre-mRNAs are remarkable. Two-thirds of the Tc1-containing introns removed from hlh-1 and unc-54 lack either the 5'-GU or AG-3' dinucleotides typically found at the termini of eukaryotic introns. We conclude that splicing to remove Tc1 from mutant pre-mRNAs allows many Tc1 insertions to be phenotypically silent. Such mRNA processing may help Tc1 escape negative selection.

Roller AB et al. (2000) Genetics "The allele-specific suppressor sup-39 alters use of cryptic splice sites in Caenorhabditis elegans."

Hoffman DC, Zahler AM, Roller AB

[Genetics, 2000]

Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 5' end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes loss of reading frame; interestingly these messages are not substrates for nonsense-mediated decay. The third splice donor, used in 10% of steady-state e936 messages, is the mutated splice donor at the wild-type position, which defines an intron beginning with UU. In the presence of a sup-39 mutation, these same three splice donors are used, but the ratio of messages produced by splicing at these sites changes. The percentage of unc-73(e936) messages containing the wild-type splice junction is increased to 33% with a corresponding increase in the level of UNC-73 protein. This sup-39-induced change was also observed when the e936 mutant intron region was inserted into a heterologous splicing reporter construct transfected into worms. Experiments with splicing reporter constructs showed that the degree of 5' splice site match to the splicing consensus sequence can strongly influence cryptic splice site choice. We propose that mutant SUP-39 is a new type of informational suppressor that alters the use of weak splice donors.

Moribe, Hiroki et al. (2019) MicroPubl Biol

Moribe, Hiroki, Mekada, Eisuke

[MicroPubl Biol, 2019]

TSP-15 is one of the 21 tetraspanins in Caenorhabditis elegans and is essential for exoskeletal (cuticle) development. A reduction in the function of TSP-15 results in a dumpy (Dpy) and/or blistered (Bli) phenotype (Moribe et al. 2004), and TSP-15 null mutants exhibit embryonic lethality (Moribe et al. 2012). sv15 is a hypomorphic allele of tsp-15, featuring a splicing error mutation in tsp-15s fourth intron. The mutation results in the immediate production of a stop codon (Fig. 1A), and the resulting truncated form of TSP-15 protein has no function. sv15 mutants are viable despite severe cuticle deficiencies since some correctly spliced transcripts are produced (Moribe et al. 2004). To further understand the functions of TSP-15 in vivo, we screened genetic suppressor mutants of tsp-15(sv15). tsp-15(sv15) worms were mutagenized with 50 mM ethyl methanesulfonate (EMS) for 4 h and F2 progeny were screened for wild-type appearance. Nine suppressor alleles (im1 to im9) were isolated from a nonclonal screen from 15,000 haploid genomes. One dominant and eight recessive suppressor alleles were included among them. The alleles were mapped by single-nucleotide polymorphism (SNP) mapping using Hawaiian variant CB4856 and characterized by DNA sequence analysis. Mutations were confirmed by complementation test, rescue by DNA transformation, and RNAi analysis. We identified one dominant allele as described here and four of eight recessive alleles, which will be described elsewhere. The dominant allele im9 was revealed to feature an intragenic mutation in intron 4 of tsp-15 (Fig. 1A). sv15/sv15im9 showed the wild-type appearance with a very mild Dpy phenotype, and the appearance of sv15im9/sv15im9 homozygotes almost matched of the wild-type. im9 mutation abolished a cryptic splice donor site, which is used in sv15, suggesting that the original splice site was possibly reused in sv15im9 mutants. To test this hypothesis, we performed quantitative RT-PCR (qRT-PCR). To detect the correct tsp-15 transcript, we designed a TaqMan MGB probe specific for the border between exons 4 and 5 (Fig. 1B). The tsp-15 primer set and TaqMan probe were designed by Primer Express software (Applied Biosystems). Total RNA and first-strand cDNA were prepared as described previously (Moribe et al. 2004). TaqMan real-time PCR was performed and analyzed using the ABI 7500 system (Applied Biosystems). RNA polymerase II subunit ama-1 was used as an internal control to normalize the tsp-15 relative value, and its primers and probe were purchased from TaqMan Gene Expression Assays (Assay ID Ce02462726_m1). The PCR reaction was performed using TaqMan Universal PCR Master Mix (Applied Biosystems), in accordance with the manufacturers protocol. Because the amplification efficiencies of tsp-15 and ama-1 were almost equal, we calculated the relative amount of tsp-15 by the comparative CT method. The relative value was determined from the mean of duplicate experiments. The results showed that the correct transcript was produced from the sv15im9 allele at a high level. sv15im9 homozygotes were comparable to tsp-15 null heterozygotes (ok881/+), which showed the wild-type appearance, in terms of the expression level of the correct transcript of tsp-15 (Fig. 1C). This suggested that the tsp-15 expression level in sv15im9 was sufficient to restore exoskeletal development. In addition, we also sequenced tsp-15 cDNA subclones isolated from sv15im9 by RT-PCR, and confirmed the presence of correctly spliced transcripts (Fig. 1D). The C. elegans splice site consensus sequences match those of vertebrates; C. elegans introns also obey the GU-AG rule (Blumenthal and Steward 1997). The splicing that occurred in sv15im9 mutants did not conform to this rule, but several instances in which the splice site did not always follow the rule have already been reported. Intron recognition involves the A+U richness of introns, which is probably significant for recognition of 5 splice sites by U1 snRNP (Blumenthal and Steward 1997).