Post-transcriptional RNA binding

Post-transcriptional control of RNA metabolism plays a major role in development and involves proteins that bind to RNA (RBPs). RBP binding has been shown to display tissue-specific activity, which when altered can result in tissue-specific mutant phenotypes. Molecular studies have identified many RBPs that bind regulatory sequences in the 3' untranslated regions of mRNAs. Studies of RBPs in C. elegans seek to provide insights into how RBPs exert coordinate control of their RNA targets and affect development.

Response to osmotic stress

The increase or decrease in solute concentrations outside of an organism can result in a stress response, which can include changes in behavior or gene expression. As an animal that is hyperosmotic to its normal environment, C. elegans exhibits structures and mechanisms to assess and deal with changes in the osmolarity of its environment. On a behavioral level, C. elegans avoids regions of high osmolarity. On a physiological level, hypertonic stress results in the upregulation of glycerol 3-phosphate dehydrogenases, gpdh-1 (strong) and gpdh-2 (weak). These genes aid in adapting to hypertonic environments by adjusting glycerol concentrations within cells.

Trans-splicing

Trans-splicing is an RNA processing event that fuses together sections of two different pre-mRNA sequences. In C. elegans, ~70% of mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders, SL1 or SL2, with more than half of all transcripts undergoing SL1 splicing. During SL1 splicing, the 5' ends of pre-mRNAs are removed and replaced with SL1 sequence in a process very closely related to cis-splicing (intron/exon processing). SL1 sequence is ~100nt and is donated by small nuclear ribonucleoprotein particles (snRNPs). The remaining genes are trans-spliced by SL2. These genes are all downstream genes in closely spaced gene clusters similar to bacterial operons. They are transcribed from a promoter at the 5' end of the cluster of between 2 and 8 genes. This transcription makes a polycistronic pre-mRNA that is co-transcriptionally processed by cleavage and polyadenylation at the 3' end of each gene, and this event is closely coupled to the SL2 trans-splicing event that occurs only ~100 nt further downstream. Recent studies on the mechanism of SL2 trans-splicing have revealed that one of the 3' end formation proteins, CstF, interacts with the only protein known to be specific to the SL2 snRNP.

Programmed cell death

Programmed cell death (PCD), or apoptosis, is an integral component of C. elegans development. During development, 131 cells are fated to die by apoptosis. PCD is easily observed with Nomarski optics in the C. elegans embryo; the nucleus of the apoptotic cell becomes refractile, resembling a flat button, making C. elegans an easy model for following apoptotic cell death. PCD in C. elegans can be divided into three main phases (with participating genes): specification of which cells should live or die (ces-1, ces-2, in general); activation of the cell killing machinery (egl-1, ced-9, ced-4 and ced-3); and an execution phase where the dying cell is dismantled and removed through phagocytosis (ced-1,-2,-5,-6,-7,-10 and-12), which occurs in concert with apoptotic DNA degradation (cps-6, nuc-1). Molecular and biochemical studies in C. elegans revealed programmed cell death mechanisms conserved in humans. The regulatory pathway that controls cell death is composed of conserved cell death activators and inhibitors: EGL-1 and BH3-domain-only proteins, CED-9 and Bcl-2, CED-4 and Apaf-1, and CED-3 and caspases, in nematodes and mammals, respectively. Further the degradation of chromosomal DNA involves a mitochondrial proapoptotic endonuclease: endonuclease G (EndoG), and AIF in mammals and their orthologs CPS-6 and WAH-1 in worms.

RNA processing

After transcription in the nucleus and before translation into a protein in the cytoplasm, newly transcribed RNA undergoes post-transcriptional modifications to become the mature mRNA. These modifications include the addition of a 5'cap and 3'poly A tail, and splicing out of noncoding introns. These modifications are needed for the RNA molecule to be protected against RNase activity as well as for it to be recognized by molecules that mediate translation into proteins. Splicing of the RNA is required to remove the portions of the message that are not supposed to be translated into the final protein product. In addition to intron and exon splicing of the pre-mRNA, ~70% of pre-mRNAs in C. elegans are trans-spliced to one of two different splice leader sequences, SL1 or SL2.

EGF signaling pathway

The Epidermal Growth Factor (EGF) signaling pathway was initially identified by Nobel Prize winner Stanley Cohen who found EGF to be involved in numerous cellular signaling events in mouse development. The single C. elegans EGF ligand, lin-3 and its receptor, lin-28 were initially found in screens for vulval development mutants. Further analysis of similar mutants, led to the identification of the C. elegans core EGFR signaling pathway components, SEM-5/GRB2, SOS-1, LET-60/RAS, LIN-45/RAF, MEK-2/MEK, and MPK-1. Positive regulators of this pathway were subsequently found in suppressor screens, and include KSR-1, SUR-8/SOC-2, and SUR-6. Negative modulators were found in further forward and reverse genetic screens and include UNC-101, SLI-1, GAP-1, SUR-5 and ARK-1 (in conjunction with GRB-2). LIN-3/EGF acts as a signal from the single anchor cell to induce neighboring undifferentiated epithelial cells to adopt the vulval fate during larval development. Impairing this signaling event results in a failure of vulval fate specification, and eventually a vulvaless animal that cannot lay eggs (the "bag of worms" phenotype). As in the mouse, EGF signaling was found to be involved in a number of other processes, although perhaps most importantly it is required for larval survival; homozygous let-23 null worms die at the L1 stage. Further, EGF signaling has been shown to function in coordinating vulva to uterus communication during development, promoting male spicule development, inducing P12.p cell fate in the epithelia, at the same time acting as a physiological signal, promoting ovulatory contractions of the gonad sheath cells, inducing a reversible nervous system quiescence during each larval molt, and regulating protein and calcium homeostasis in the adult worm.

Germline apoptosis

Germline apoptosis is an integral part of oogenesis in many animals, including humans. Germline apoptosis relies on key components of the core apoptotic machinery, including CED-3 and CED-4 and on the same engulfment mechanism as for somatic corpses. The Ras pathway might directly or indirectly regulate germline PCD as shown, unlike somatic apoptosis, cell death in the germline is not developmentally programmed by cell lineage. Germline apoptosis can be divided into two categories, physiological or stress-induced. Physiological germline apoptosis reduces the number of cells that complete oogenesis, and its induction is independent of the BH3-only apoptosis effecter EGL-1, differing from other apoptotic pathways. Stress-induced germline apoptosis is triggered by a genomic integrity checkpoint. In addition, exposure to certain environmental insults or pathogens induces germ cell apoptosis. Germ cell apoptosis in C. elegans occurs only during oocyte production, and is further restricted to the gonadal loop region, where developing oocytes exit the pachytene stage of meiotic prophase and transition into diplotene. let-60 (ras), lin-45 (raf), mek-2 (MAPKK), and mpk-1 (MAPK) have been show to be required for exit from pachytene arrest, as well as for PCD in the female germline, suggesting the Ras pathway might directly or indirectly regulate germline PCD.