Kotagama, Kasuen et al. (2021) International Worm Meeting "Studying the tissue-specific functions of the conserved mir-51 family of microRNAs"

McJunkin, Katherine, Grimme, Acadia, Kotagama, Kasuen

[International Worm Meeting, 2021]

MicroRNA (miRNA) are small non-coding RNAs that regulate gene expression post-transcriptionally by targeting mRNAs for translational inhibition and degradation. MiRNAs are functionally categorized into families based on nucleotides 2-7 at their 5' ends, which is referred to as the "seed" region. Due to the length of the seed sequence, a single miRNA is capable of regulating many target transcripts, and identifying functional miRNA targets can be difficult. Members of a miRNA family share the same seed sequence and are believed to have largely overlapping targets. The mir-51/100 family of miRNAs is widely conserved across metazoans and is essential for embryonic development in C. elegans. In the worm, this miRNA family consists of six mature miRNAs, mir-51 through mir-56, and loss of the entire mir-51 family leads to embryonic lethality with a pharynx unattached phenotype. Experiments with extrachromosomal transgenes indicate that the mir-51 family members are functionally redundant when rescuing the embryonic lethality. The redundancy between family members indicates that the mir-51 family probably shares a core set of essential targets; however a comprehensive list of the essential targets regulated by the mir-51 family remains elusive. The mir-51 family is expressed beyond embryonic development, and hypomorphic mir-51 family mutants that bypass embryonic lethality display post-embryonic phenotypes, which include developmental delays, reduced brood size, and food avoidance, suggesting that the mir-51 family has functions beyond embryonic development. In this study, we will attempt to identify the post-embryonic roles of the mir-51 family using tissue-specific techniques. We have generated transgenic strains expressing mir-52 tissue-specifically in the intestine, body wall muscle, pharynx and neurons in a mir-51 family hypomorphic background and are testing these strains for rescue of post-embryonic phenotypes. By combining these tissue-specific requirements with bioinformatic predictions of miRNA targets and tissue-specific transcriptome data, we will identify candidate miRNA targets. We will then use CRISPR-Cas9 to perform deletions in the predicted miRNA binding sites of these candidate genes and attempt to recapitulate the post-embryonic phenotypes caused by deficiency of the mir-51 family. The overall goal of this study is to identify the essential targets of this conserved miRNA family and to investigate the significance of these interactions for development.

Kotagama, Kasuen et al. (2019) International Worm Meeting "ALG-1 influences accurate mRNA splicing patterns in the C. elegans intestine and body muscle tissues by modulating splicing factor activities."

Schorr, Anna, Steber, Hannah, Kotagama, Kasuen, Mangone, Marco

[International Worm Meeting, 2019]

MicroRNAs (miRNAs) are known to modulate gene expression, but their activity at the tissue-specific level remains largely uncharacterized. In order to study their contribution to tissue-specific gene expression, we developed novel tools to profile miRNA targets in the C. elegans intestine and body muscle. We validated many previously described interactions, and identified ~3,500 novel targets. Many of the miRNA targets curated are known to modulate the functions of their respective tissues. Within our datasets we observed a disparity in the usage of miRNA-based gene regulation between the intestine and body muscle. The intestine contained significantly more miRNA targets than the body muscle highlighting its transcriptional complexity. We detected an unexpected enrichment of RNA binding proteins targeted by miRNA in both tissues, with a notable abundance of RNA splicing factors. We developed in vivo genetic tools to validate and further study three mRNA splicing factors identified as miRNA targets in our study (asd-2, hrp-2 and smu-2), and show that these factors indeed contain functional regulatory elements in their 3'UTRs that are able to repress their expression in the intestine. In addition, the alternative splicing pattern of their respective downstream targets (unc-60, unc-52, lin-10 and ret-1) is dysregulated when the miRNA pathway is disrupted. A re-annotation of the transcriptome data in C. elegans strains that are deficient in the miRNA pathway from past studies supports and expands on our results. This study highlights an unexpected role for miRNAs in modulating tissue-specific gene isoforms, where post-transcriptional regulation of RNA splicing factors associates with tissue-specific alternative splicing.