Post-transcriptional regulation of the central apoptotic pathway by microRNAs and RNA-binding proteins during C. elegans development

Sherrard, Ryan W. (2019): Post-transcriptional regulation of the central apoptotic pathway by microRNAs and RNA-binding proteins during C. elegans development. Dissertation, LMU München: Fakultät für Biologie

Abstract

Programmed cell death is an essential process during animal development. One type of cell death—apoptosis—is well understood at the molecular level, in large part due to genetic studies in the nematode Caenorhabditis elegans. The central apoptotic pathway in C. elegans consists of the four conserved genes egl-1 (BH3-only), ced-9 (Bcl-2), ced-4 (Apaf-1), and ced-3 (caspase), which act in a linear pathway. It is crucial that the activity of this pathway only triggers apoptosis in those cells programmed to die, and this requires regulation at multiple levels of gene expression. Although several studies have examined this regulation, post-transcriptional control of the pathway is still not well characterized. Here, I investigate regulation of the central apoptotic pathway by two prominent post- transcriptional mechanisms: microRNAs (miRNAs) and RNA-binding proteins (RBPs). First, I present evidence that the miR-35 family and the miR-58 bantam family of miRNAs directly target conserved elements in the 3ʹUTR of egl-1 mRNA and act cooperatively to repress its expression. This repression is crucial during embryogenesis, as loss of the mir-35 family leads to the inappropriate death of cells that are not programmed to die, and this phenotype is enhanced by the additional loss of the mir-58 family. These inappropriately dying cells are exclusively mothers and sisters of cells that are programmed to die, and their precocious and collateral deaths result in the formation of abnormally large cell corpses. Using single-molecule RNA FISH, I show that egl-1 is already transcribed in mother cells, and that both miR-35- and miR-58-family miRNAs function to maintain the copy number of egl-1 mRNA below a critical threshold—failure to do so results in precocious death of the mother cell. Furthermore, it seems that these two miRNA families are not required for the turnover of egl-1 mRNA over time in the daughter that survives. Considering that egl-1 transcription is controlled by numerous factors with varying modes of activity, the cooperative activity of miR-35- and miR-58- family miRNAs may buffer any lineage-specific differences in egl-1 transcription, thereby ensuring EGL-1 activity only reaches a level sufficient to trigger death in daughter cells that are programmed to die. Next, I describe a novel cell-death role for the gene puf-8, which encodes a conserved member of the Pumilio/FBF (PUF) family of RBPs. I show that animals lacking puf-8 exhibit two contrasting cell-death abnormalities. First, some cells die inappropriately, and these cells belong to both cell-death and non-cell-death lineages. Second, programmed cell death is delayed during the first wave of embryonic cell death. These abnormalities are not present upon nor enhanced by the loss of closely related puf-9, nor is the phenotype present upon knockdown of another member of the PUF family, fbf; however, fbf knockdown does suppress the large-cell-corpse phenotype in mir-35-family mutants. All four genes of the central apoptotic pathway harbor PUF-8-binding elements (PBEs) and/or FBF-binding elements (FBEs) in their 3ʹUTRs, and two FBEs in egl-1 can mediate the repression of a transgenic reporter. Therefore, puf-8 and fbf exhibit activity that both promotes and suppresses the cell-death pathway, and their prevailing activity might be regulated in a tissue- or cell-specific manner. Taken together, these findings show complex regulation of the central apoptotic pathway by both miRNAs and RBPs, and mounting evidence in the field suggests these two mechanisms can function cooperatively in the regulation of common targets. Evidence of cell-death genes being targeted by miRNAs and RBPs in mammals supports the possibility of this regulation being conserved in higher animals, which could have implications for the medical field. Our understanding of programmed cell death and its regulation has already led to the development of drugs that trigger apoptosis in cancer cells, and furthering this understanding could aid in the development of novel disease treatments.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: programmed cell death, apoptosis, development, microRNAs, RBPs, C. elegans
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 21. Februar 2019
• 1. Bericht­erstatter:in: Conradt, Barbara