Among the many non-protein coding RNAs that have been discovered, small (s)RNAs that are ~18-30 nucleotides in length play important biological roles in diverse pathways collectively called RNA interference (RNAi). These biological roles range from proper cell division and maintenance of cellular health to organismal development and fighting disease and infection. At present, the Lee Lab is keenly interested in understanding the molecular mechanisms by which long RNA precursors are recognized for sRNA production, and the cellular processes that require sRNAs and/or RNAi protein machinery. For our studies, we employ the single-celled organism Tetrahymena thermophila as a model system which offers much potential for uncovering aspects of foundational biology that are broadly conserved, as well as fascinating points of biological diversity.
RNA interference (RNAi) is a biological process used by many eukaryotes as a defense mechanism against viruses and mobile elements, like transposons, and to repress gene expression. RNAi pathways play a variety of other important roles in diverse eukaryotic cells, including centromere function in chromosome segregation and genome organization.
Despite the diversity of biological functions that RNAi pathways serve, they are unified in their production of sRNAs. Depending on the organism, a variety of proteins serve to generate and interact with these sRNAs, including RNA-dependent RNA Polymerases (RdRPs), Dicers, and Argonaute/Piwi (Ago/Piwi) proteins. RdRPs act in RNA-dependent RNA Polymerase complexes (RDRCs) to turn the sRNA precursor transcripts into double stranded (ds)RNA or use target transcripts as templates to generate short sRNAs directly. Dicers cut long dsRNA RdRC products up into sRNA-length products. SRNAs ~18-35 nucleotides (nts) are then assembled with Ago/Piwi family proteins for downstream activities in gene silencing.
Conserved RNAi machinery in eukaryotes showing the biogenesis of sRNAs from long RNA precursor transcripts. In some systems, RdRPs directly synthesize sRNAs from transcripts without involvement of a Dicer. Figure created with BioRender.com by Maya Matsumoto.
Because of the ability of RNAi to selectively suppress genes, it is often used in research studies dissecting biological roles of particular genes and has found applications in therapeutic medicines. As a major gene regulatory pathway, endogenous RNAi pathways play important roles in many biological processes that impact human health, including organism development, the development of cancers, and combating viral infections.
Eukaryotic supergroups and subgroups as related to Tetrahymena with the eukaryotic root shown on the left. Figure from Ruehle et al. (2016) adapted from Lynch et al. (2014) using the National Center for Biotechnology Information taxonomy browser. Fluorescence microscopy image adapted from Wloga and Gaertig, as in Goldfarb and Gorovsky (2009), shows the MIC and MAC in blue and cilia in green.
Our lab uses as an experimental model system the ciliate Tetrahymena thermophila to study RNAi. Tetrahymena are single-celled protozoans descended from an early branching lineage of eukaryotes. As such, they possess many evolutionarily conserved eukaryotic processes, some of which have been lost in other single-celled model organisms, and also offer the opportunity to uncover previously unknown biological diversity.
Like many ciliates, Tetrahymena exhibit nuclear dimorphism, meaning they have two nuclei: the diploid germline micronucleus (MIC) and polyploid somatic macronucleus (MAC). The MAC is the transcriptionally active nucleus, while the MIC is primarily transcriptionally silent, undergoes meiosis during conjugation (sexual reproduction), and is the source genetic material for new MACs and MICs that end up in progeny cells.
As an experimental model system, Tetrahymena's strengths lie in its fast doubling times, requirement for minimal and inexpensive media for growth, fully sequenced genome, relatively large size and optically clear properties, and ease of genetic disruption, making it amenable to studies employing biochemistry, microscopy, bioinformatics, and reverse genetics.
Tetrahymena also have a long history of contributing to major insights in molecular biology that are applicable to a broad variety of eukaryotes, including humans. These include the identification of histone variants and histone modification differences in heterochromatin and euchromatin, recognition of checkpoint-monitored processes in mitosis and meiosis, discovery of catalytic RNAs, RNA-mediated transgenerational inheritance, and recognition of telomeres and telomerase.
Two RNAi pathways have been discovered in Tetrahymena. The first is well studied and assists in developing a new macronucleus (MAC) from the zygotic micronucleus (MIC) during conjugation (Rzeszutek, 2020).
Our lab focuses on a second, less well-understood RNAi pathway in Tetrahymena that generates ~23-24 nucleotide (nt) sRNAs. Our research has shown that this pathway processes diverse sRNA precursor transcripts into ~23-24 nt sRNAs through the employment of three distinct RDRCs, each containing an RdRP called Rdr1, that physically and functionally couple to the Dicer enzyme Dcr2 (Lee and Collins, 2006) (Lee, Talsky, and Collins, 2009). These ~23-24 nt sRNAs are primarily bound by the Piwi homolog proteins Twi 2, 7, or 8 for yet unknown downstream functions (Couvillion et al., 2009).
RNAi biogenesis pathway in Tetrahymena producing ~23-24 nt sRNAs. Figure created with BioRender.com by Maya Matsumoto.
Tetrahymena-specific RNAi pathway labeled with the Lee Lab’s key research questions. Figure created with BioRender.com by Maya Matsumoto.
The Lee Lab is interested in these key questions relating to RNAi in Tetrahymena:
1. What are the biological impacts of these RNAi pathways?
Our first insights into this question have recently been published! Check out our publication in Molecular Biology of the Cell for a look at our findings.
2. What does Rsp1 (RNA-silencing protein) do molecularly?
Previous work has shown that Rsp1 is required for the accumulation of all RDRC-dependent sRNAs, yet we still don't understand the molecular reason for this requirement (Talsky and Collins, 2012). Current work in the Lee Lab is focused on pinpointing where in the pathway it functions.
3. How are precursor transcripts recognized for sRNA processing?
Distinct classes of putative precursors exist in Tetrahymena, yet we still don't have a handle on what fates a particular transcript for recognition by RDRCs and Dcr2. Identification of these determinants is ongoing in the Lee Lab.
4. Are there alternative pathways that target precursor transcripts?
Certain precursor transcripts have mRNA-like features, the significance of which the Lee Lab is currently investigating.
The Lee Lab uses a variety of experimental approaches to tackle our unanswered research questions. These approaches include reverse genetics, microscopy (including fluorescence microscopy), image analysis using coding, bioinformatics, and biochemistry.
Couvillion, MT, et al. Sequence, biogenesis, and function of diverse small RNA classes bound to the Piwi family proteins of Tetrahymena thermophila. Genes Dev 23.17, 2016-32 (2009). https://doi.org/10.1101/gad.1821209
Goldfarb, DS, Gorovsky, MA. Nuclear dimorphism: two peas in a pod. Current Biology 19.11, PR449-R452 (2009). https://doi.org/10.1016/j.cub.2009.04.023
Gutbrod, MJ, Martienssen, RA. Conserved chromosomal functions of RNA interference. Nat Rev Genet 21, 311–331 (2020). https://doi.org/10.1038/s41576-019-0203-6
Lee, SR, Collins, K. Two classes of endogenous small RNAs in Tetrahymena thermophila. Genes Dev 20.1, 28-33 (2006). https://doi.org/10.1101/gad.1377006
Lee, SR, Talsky, KB, Collins, K. A single RNA-dependent RNA polymerase assembles with mutually exclusive nucleotidyl transferase subunits to direct different pathways of small RNA biogenesis. RNA 15.7, 1363-74 (2009). https://doi.org/10.1261/rna.1630309
Lee, SR. Disruption of a ~23-24 nucleotide small RNA pathway elevates DNA damage responses in Tetrahymena thermophila. Molecular Biology of the Cell (2021). https://www.molbiolcell.org/doi/abs/10.1091/mbc.E20-10-0631
Ruehle, MD, Orias E, Pearson C. Tetrahymena as a unicellular model eukaryote: genetic and genomic tools. Genetics 203.2, 649-655 (2016). https://doi.org/10.1534/genetics.114.169748
Rzeszutek, I, Maurer-Alcala, XX, Nowacki, M. Programmed genome rearrangements in ciliates. Cell Mol Life Sci 77.22, 4615-4629 (2020). https://doi.org/10.1007/s00018-020-03555-2
Talsky, KB, Collins, K. trand-asymmetric endogenous Tetrahymena small RNA production requires a previously uncharacterized uridylyltransferase protein partner. RNA 18.8, 1553-62. https://doi.org/10.1261/rna.033530.112