Antisense RNA and RNAi in protozoan parasites: Working hard or hardly working?

doi:10.1016/j.molbiopara.2007.10.004

Molecular and Biochemical Parasitology

Volume 157, Issue 2, February 2008, Pages 117-126

https://doi.org/10.1016/j.molbiopara.2007.10.004 Get rights and content

Abstract

The complex life cycles of many protozoan parasites require the ability to respond to environmental and developmental cues through regulated gene expression. Traditionally, parasitologists have investigated these mechanisms by identifying and characterizing proteins that are necessary for the regulated expression of the genetic material. Although often successful, it is clear that protein-mediated gene regulation is only part of a complex story in which RNA itself is endowed with regulatory functions. Herein, we review both the known and potential regulatory roles of two types of RNA pathways within protozoan parasites: the RNA interference pathway and natural antisense transcripts. A better understanding of the native role of these pathways will not only enhance our understanding of the biology of these organisms but also aid in the development of more robust tools for reverse genetic analysis in this post-genomic era.

Introduction

Long known for its critical roles in the storage and expression of genetic material, RNA is now thought to be involved in an ever-increasing myriad of cellular functions. In the last 5–10 years, it is has become evident that RNA can also directly regulate many cellular processes in eukaryotes including transcription, RNA stability, translation, retrotransposition, DNA replication and DNA packaging. Since protozoan parasites have complex life cycles involving multiple hosts and changing environments, it is likely that RNA may have a similarly diverse regulatory role in these organisms. In fact, RNA-mediated regulation may be particularly relevant to this group of organisms since gene expression does not always follow standard eukaryotic paradigms. Further, interesting and unusual RNA biology has typically been the rule in protozoan parasites starting with the discoveries of RNA editing and trans-splicing in trypanosomatids [1], [2]. In this article, we will review two types of RNA pathways found in protozoan parasites: RNA interference (RNAi) and natural antisense transcripts (NATs). An emphasis is placed on recent developments in the field of RNAi, and readers are referred to previous reviews on the subject [3], [4]. Although the study of these RNA pathways is still in its infancy in most protozoan parasites, these pathways will be discussed with respect to their potential to regulate gene expression and use as powerful tools for genetic analysis.

Section snippets

Overview of RNAi in eukaryotes: endogenous regulatory mechanism and powerful reverse genetics tool

RNAi is the process by which a specific double-stranded RNA is cleaved into short interfering RNAs (siRNAs), and the siRNAs guide the RNase-mediated cleavage of the homologous target mRNA. The mechanism of RNAi and its relevant biology has received an enormous amount of attention since the original descriptive report by Fire et al. in 1998 [5] (Fig. 1), and exhaustive work has led to the identification of essential enzymatic components. The cleavage of double-stranded RNA is catalyzed by the

Comparative biology reveals RNAi machinery in only a subset of protozoan parasites

The phenomenon of RNAi in protozoan parasites was first reported from studies of Trypanosoma brucei in the Ullu laboratory in 1998 [11], and has been previously reviewed [4]. The T. brucei RNAi pathway has many of the characteristic features of known RNAi pathways in model organisms including the generation of siRNA [12], [13], and required Argonaute and Dicer genes [14], [15], [16], [17]. The initial work in T. brucei urged others to begin investigating which other protozoan parasites contain

Functional RNAi studies in protozoan parasites: some clarity, more controversy

The strongest strategy to determine if protozoan parasites have an active RNAi pathway is a functional approach. Long double-stranded RNAs and siRNAs have been used to silence expression of endogenous transcripts, and effectiveness has been measured by the loss of the endogenous transcripts. Nonetheless, one weakness of this strategy is the difficulty in correlating a positive result with the presence of an RNAi pathway dependent upon double-stranded RNA substrates, siRNAs, and transcript

Intrinsic cellular roles for RNAi machinery in protozoan parasites?

The RNAi machinery, where present and functional, potentially plays an important role in the biology of these protozoan parasites. Evidence for a role for RNAi in parasite biology primarily comes from experiments in T. brucei, which suggest that the RNAi pathway may have several endogenous roles. First, T. brucei parasites deficient in RNAi have higher levels of the full length retrotransposon transcripts SLACS and Ingi, and less SLACS and Ingi siRNAs [14], [16], [39], [40]. Thus, the function

Stuck in first gear: RNAi as a genetics tool in protozoan parasites

Although there have been functional studies suggesting the presence of RNAi-like pathways in several protozoan parasites, RNAi has not been adopted as a reliable and widespread strategy for reverse genetics in protozoan parasites except T. brucei. Why is this? At the moment, the case for the group II organisms seems relatively simple, as they seem to lack the RNAi machinery. But what about group III organisms, especially T. gondii and Plasmodium spp., in which there is either bioinformatic or

Reconstituting an active RNAi pathway: far-fetched idea or far-reaching implications?

With the successful use of RNAi in T. brucei, using genetic manipulation to create active RNAi pathways in organisms where it is lacking presents an appealing, albeit potentially unattainable, possibility. In T. brucei, the distantly related human Ago2 was shown to complement TbAgo1, providing the first demonstration that Argonaute proteins could be interchanged and function in distantly related organisms [66]. Additionally, Rivas et al. report that recombinant human Ago2 can combine with a

Natural antisense transcripts in protozoan parasites: a universal phenomenon?

Antisense RNA transcripts are complimentary to coding transcripts and have been discovered in numerous organisms including bacteria [67], plants [68], [69], mice [70] and humans [71], [72]. Because they are a fundamental component of many transcriptomes, the term natural antisense transcripts is used to differentiate these molecules from engineered antisense RNAs. In the past several years, NATs have been detected in the transcriptomes of many protozoan parasite including P. falciparum [73],

What is the function of NATs in protozoan parasites?

The function of NATs in protozoan parasites has largely remained an enigma. Although NATs in some organisms such as humans contain open reading frames [92], the majority of NATs examined in protozoan parasites does not contain obvious open reading frames and are predicted to be non-coding RNAs. However, exceptions to this model exist [77], and only an analysis of more cDNA sequences would shed light on the coding potential of NATs. A role for protozoan NATs in gene expression represents an

Do NATs generate substrates for RNAi?

Another process that needs further investigation is the potential relationship between NATs and the RNAi pathway. Do NATs basepair with sense transcripts in vivo and, if so, does this lead to the generation of double-stranded RNA substrates for the RNAi pathway, in species where it is present (see above)? This event depends on both RNAs being present in the same cellular compartment at the same time and annealing. In the original RNAi description in T. brucei, exogenous antisense α-tubulin RNA

Genetic potential of antisense RNA inhibition in protozoan parasites

Engineered antisense RNA inhibition could be an excellent tool for reverse genetic analysis in protozoan parasites. Antisense RNA inhibition has many advantages over conventional gene knockout strategies, some of which are shared with RNAi. For example, antisense RNA and RNAi both allow for the incomplete elimination of protein production and thus analysis of essential loci. Both strategies are not strictly dependent upon plasmid integration into genomic DNA, which is still difficult and timely

Conclusions

With the recent completion of several genome-sequencing projects, we are currently at a turning point in our understanding of protozoan parasites. However, many pressing biological and technical questions still remain which include understanding the regulation of gene expression in these organisms as well as the development of more robust tools for rapid functional analysis. A common answer to both of these unknowns may exist in the endogenous regulatory RNA pathways that exist in these

Acknowledgements

We thank Robert D. Simon, Jenna Tabor-Godwin, Devin Chandler-Militello, Tiffany DeSimone and Bradley Coleman for critical reading of this manuscript.

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ReviewAntisense RNA and RNAi in protozoan parasites: Working hard or hardly working?

Abstract

Introduction

Section snippets

Overview of RNAi in eukaryotes: endogenous regulatory mechanism and powerful reverse genetics tool

Comparative biology reveals RNAi machinery in only a subset of protozoan parasites

Functional RNAi studies in protozoan parasites: some clarity, more controversy

Intrinsic cellular roles for RNAi machinery in protozoan parasites?

Stuck in first gear: RNAi as a genetics tool in protozoan parasites

Reconstituting an active RNAi pathway: far-fetched idea or far-reaching implications?

Natural antisense transcripts in protozoan parasites: a universal phenomenon?

What is the function of NATs in protozoan parasites?

Do NATs generate substrates for RNAi?

Genetic potential of antisense RNA inhibition in protozoan parasites

Conclusions

Acknowledgements

Cell

Gene

Protist

Structure

J Biol Chem

Exp Parasitol

Mol Biochem Parasitol

Mol Biochem Parasitol

Mol Biochem Parasitol

Mol Biochem Parasitol

Biochem Biophys Res Commun

Biochem Biophys Res Commun

J Biol Chem

Mol Biochem Parasitol

Biochem Biophys Res Commun

J Mol Biol

FEBS Lett

Mol Biochem Parasitol

Exp Parasitol

Mol Biochem Parasitol

Mol Biochem Parasitol

Cell

Mol Biochem Parasitol

Mol Biochem Parasitol

J Biol Chem

Mol Biochem Parasitol

Trends Genet

Mol Biochem Parasitol

Mol Biochem Parasitol

Int J Parasitol

J Biol Chem

Cell

Cell

Cell

Cell

Mol Biochem Parasitol

RNA interference in protozoan parasites

Cell Microbiol

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Nature

Role for a bidentate ribonuclease in the initiation step of RNA interference

Nature

Purified Argonaute2 and an siRNA form recombinant human RISC

Nat Struct Mol Biol

On the origin and functions of RNA-mediated silencing: from protists to man

Curr Genet

Revealing the world of RNA interference

Nature

Double-stranded RNA induces mRNA degradation in Trypanosoma brucei

Proc Natl Acad Sci USA

RNA interference in Trypanosoma brucei: cloning of small interfering RNAs provides evidence for retroposon-derived 24-26-nucleotide RNAs

RNA

An siRNA ribonucleoprotein is found associated with polyribosomes in Trypanosoma brucei

RNA

An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei

RNA

Argonaute protein in the early divergent eukaryote Trypanosoma brucei: control of small interfering RNA accumulation and retroposon transcript abundance

Mol Cell Biol

TbAGO1, an argonaute protein required for RNA interference, is involved in mitosis and chromosome segregation in Trypanosoma brucei

BMC Biol

Comparative genomic analysis of three Leishmania species that cause diverse human disease

Nat Genet

Structural basis for double-stranded RNA processing by Dicer

Science

Review
Antisense RNA and RNAi in protozoan parasites: Working hard or hardly working?