Kniss SL et al. (2000) Midwest Worm Meeting "Genetic analysis of C. elegans elongation using sma-1 suppressors"

Kniss SL, Austin JA

[Midwest Worm Meeting, 2000]

Elongation of the C. elegans embryo occurs as the result of changes in cell shape that occur during a three hour period midway through embryogenesis. Previous work has shown that these cell shape changes are dependent on circumferential actin bundles in the embryonic epidermal cells. It is believed that contraction of these bundles generates the force used to elongate the embryo. Normal elongation also requires SMA-1 beta-spectrin, a component of the apical membrane cytoskeleton; animals lacking SMA-1 fail to elongate during embryogenesis and are shorter at hatching than wild type worms. Results from our lab indicate that SMA-1 is required both for attachment of the actin bundles to the apical epidermal membrane and for the efficient conversion of contractile force generated by the actin cytoskeleton into changes in cell shape (V. Praitis, unpublished data.) In order to identify proteins that act with SMA-1 during elongation of the embryo, we have isolated suppressors of the sma-1 phenotype. In addition to partial suppression of the sma-1 defect in elongation, we have found that the suppressed animals also have phenotypes, such as constitutive egg laying (Egl-c) and hyperactive body movement, that indicate an increase in muscle contraction. Based on the phenotypes of the suppressed animals, we hypothesize that these mutations affect proteins that regulate both cytoskeletal and muscle contraction. If so, suppression of the sma-1 elongation defect may be due to an increase in the contractile force generated by the epidermal actin fibers. We have mapped several of the sma-1 suppressors using their Egl-c phenotype and are in the process of cloning the genes containing these mutations. Identifying the normal function of these suppressor genes should reveal parallels between cytoskeletal and muscle contraction and increase our understanding of the mechanisms that regulate the elongation of the C. elegans embryo.

Kniss SL et al. (1998) Midwest Worm Meeting "A genetic analysis of C. elegans elongation using sma-1 suppressors"

Austin JA, Kniss SL

[Midwest Worm Meeting, 1998]

During morphogenesis, the <EM>C. elegans</EM> embryo elongates four-fold from a ball of cells into a long, thin worm. This elongation is dependent on circumferentially oriented actin bundles which form in the embryonic epidermal cells and are thought to generate the contractile force required for elongation. SMA-1 is a cytoskeletal protein which is homologous to <EM>Drosophila</EM> Beta-H-spectrin and is required for normal elongation: in <EM>sma-1</EM> mutants, the rate of elongation is decreased resulting in larvae that are shorter than wild-type at hatching. In many of the <EM>sma-1</EM> mutants it is been found that the organization of the epidermal actin bundles is altered (V. Praitis, personal communication). The <EM>sma-1</EM> alleles isolated in our lab vary in their effect on the rate of elongation suggesting that, in addition to its likely structural function, SMA-1 plays a role in regulation of elongation. To understand these functions of SMA-1, we have begun to isolate suppressors of <EM>sma-1</EM> mutants. We have designed a simple genetic screen that takes advantage of the inability of <EM>sma-1</EM> larvae to chemotax to food and have used this screen to isolate both dominant and recessive suppressors. We are currently examining the suppressed phenotypes and mapping the mutations. Characterization of our <EM>sma-1</EM> suppressors will begin to answer questions regarding the role of the cytoskeleton during morphogenesis and the function of SMA-1 in the regulation of elongation. In addition, we hope to understand the relationship between SMA-1's role in body elongation and its role in morphogenesis of the pharynx and excretory cell canal.

SL Kniss et al. (1999) International C. elegans Meeting "Genetic analysis of C. elegans elongation using sma-1 suppressors"

SL Kniss, J Austin

[International C. elegans Meeting, 1999]

SMA-1 spectrin is required for changes in cell shape that occur as the C. elegans embryo elongates from a ball of cells into a long, thin worm. Animals lacking SMA-1 are shorter at hatching than wild type animals due to a decreased rate of embryonic elongation. Normal elongation depends on circumferential actin bundles in the embryonic epidermal cells; organization of these bundles is disrupted in sma-1 mutants suggesting that SMA-1 secures the actin bundles to the apical membrane during elongation (V. Praitis, unpublished results). The sma-1 alleles isolated in our lab vary in their effect on elongation rate suggesting that SMA-1 may play a regulatory as well as a structural role during elongation. To identify proteins that act with SMA-1 during morphogenesis, we isolated phenotypic suppressors of the sma-1 elongation defect. We have identified both dominant and recessive suppressors using a screen based on the inability of sma-1 L1 larvae to chemotax to food. The suppressing mutations vary in their effect on elongation as well as other phenotypic characteristics including viability, egg laying and mobility. We are currently generating loss-of-function alleles of the genes containing dominant suppressor mutations in order to map and clone these genes. There are several mechanisms by which mutations in the suppressed animals may result in increased hatchling length. First, suppressing mutations may strengthen a weak association between SMA-1 and an interacting protein such as protein 4.1 or a -spectrin. Second, proteins that act in parallel to SMA-1 to link the actin cytoskeleton to the plasma membrane may be overexpressed allowing them to substitute for defective SMA-1 in the mutants. Third, suppressing mutations may alter the strength of contraction of the actin fibers. Finally, our mutations may increase the stability of sma-1 mRNA since the sma-1 allele used in our screens is suppressible by smg mutations. To distinguish between these possibilities, we are comparing the organization of the actin bundles in our suppressed mutants to that in the original sma-1 mutant. By examining suppressor phenotypes and identifying the genes containing suppressing mutations, we hope to understand the role of these genes in C. elegans morphogenesis.

Nilsen TW et al. (1989) Mol Cell Biol "Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum."

Shambaugh JD, Putnam L, Faser C, Denker JA, Bennett KL, Chubb G, Nilsen TW

[Mol Cell Biol, 1989]

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.

Van Doren K et al. (1988) Nature "Trans-spliced leader RNA exists as small nuclear ribonucleoprotein particles in Caenorhabditis elegans."

Van Doren K, Hirsh DI

[Nature, 1988]

Maturation of some messenger RNAs in the nematode Caenorhabditis elegans involves the acquisition of a 22-base leader at their 5' ends. This 22-base leader, called the spliced leader (SL), is derived from the 5' end of a precursor RNA of 90-100 bases, called spliced leader RNA (SL RNA). SL RNA is transcribed from a 1-kilobase DNA repeat which also encodes the 5S ribosomal RNA. A subset of mRNAs in C. elegans acquire SL from SL RNA by a trans-splicing mechanism. SL behaves as a 5' exon in the trans-splicing reaction. Using antisera against the Sm antigen that is associated with small nuclear ribonucleoprotein particles (snRNPs), we precipitated SL RNA from extracts of C. elegans, indicating that it is bound by the Sm antigen in vivo. SL RNA also possesses the unique trimethylguanosine (m32,2,7G) cap characteristic of most small nuclear RNAs. Therefore, SL RNA is a chimaeric molecule, made up of an snRNA attached to a 5' exon and is a constituent of a snRNP.

Denker JA et al. (1996) RNA "Multiple requirements for nematode spliced leader RNP function in trans-splicing."

Yu YT, Denker JA, Kanost RA, Nilsen TW, Maroney PA

[RNA, 1996]

The 5'' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5'' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5'' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5'' splice site in SL addition trans-splicing.

Barnes SN et al. (2019) Sci Rep "Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1."

Seetharam A, Maier TR, Barnes SN, Severin AJ, Baum TJ, Masonbrink RE, Sindhu AS

[Sci Rep, 2019]

Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5'-ends of transcripts during RNA-seq library preparation, known as 5'-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences.

Yague-Sanz C et al. (2018) Gigascience "SL-quant: A fast and flexible pipeline to quantify spliced leader trans-splicing events from RNA-seq data."

Yague-Sanz C, Hermand D

[Gigascience, 2018]

Background: The spliceosomal transfer of a short spliced leader (SL) RNA to an independent pre-mRNA molecule is called SL trans-splicing and is widespread in the nematode C. elegans. While RNA-seq data contain information on such events, properly documented methods to extract them are lacking. Findings: To address this, we developed SL-quant, a fast and flexible pipeline that adapts to paired-end and single-end RNA-seq data and accurately quantifies SL trans-splicing events. It is designed to work downstream of read mapping and uses the reads left unmapped as primary input. Briefly, the SL-sequences are identified with high specificity and are trimmed from the input reads, which are then re-mapped on the reference genome and quantified at the nucleotide position level (SL trans-splice sites) or at the gene level. Conclusions: SL-quant completes within 10 minutes on a basic desktop computer for typical C.elegans RNA-seq datasets, and can be applied to other species as well. Validating the method, the SL trans-splice sites identified display the expected consensus sequence and the results of the gene-level quantification are predictive of the gene position within operons. We also compared SL-quant to a recently published SL-containing read identification strategy which revealed being more sensitive, but less specific than SL-quant. Both methods are implemented as a bash script available under the MIT licence at https://github.com/cyaguesa/SL-quant. Full instructions for its installation, usage, and adaptation to other organisms are provided.

Denker JA et al. (2002) Nature "New components of the spliced leader RNP required for nematode trans-splicing."

Denker JA, Maroney PA, Nilsen TW, Zuckerman DM

[Nature, 2002]

Pre-messenger-RNA maturation in nematodes and in several other lower eukaryotic phyla involves spliced leader (SL) addition trans-splicing. In this unusual RNA processing reaction, a short common 5'' exon, the SL, is affixed to the 5''-most exon of multiple pre-mRNAs. The nematode SL is derived from a trans-splicing-specific approximately 100-nucleotide RNA (SL RNA) that bears striking similarities to the cis-spliceosomal U small nuclear RNAs U1, U2, U4 and U5 (refs 3, 4); for example, the SL RNA functions only if it is assembled into an Sm small nuclear ribonucleoprotein (snRNP). Here we have purified and characterized the SL RNP and show that it contains two proteins (relative molecular masses 175,000 and 30,000 (M(r) 175K and 30K)) in addition to core Sm proteins. Immunodepletion and reconstitution with recombinant proteins demonstrates that both proteins are essential for SL trans-splicing; however, neither protein is required either for conventional cis-splicing or for bimolecular (trans-) splicing of fragmented cis constructs. The M(r) 175K and 30K SL RNP proteins are the first factors identified that are involved uniquely in SL trans-splicing. Several lines of evidence indicate that the SL RNP proteins function by participating in a trans-splicing specific network of protein protein interactions analogous to the U1 snRNP SF1/BBP U2AF complex that comprises the cross-intron bridge in cis-splicing.

Bektesh SL et al. (1988) Nucleic Acids Res "C. elegans mRNAs acquire a spliced leader through a trans-splicing mechanism."

Hirsh DI, Bektesh SL

[Nucleic Acids Res, 1988]

A 22 nt spliced leader (SL) is added to some C. elegans mRNAs by a process of discontinuous RNA synthesis. The SL is synthesized as part of a larger precursor RNA, the leader RNA (LR) which is about 100 nt long.