Cells control RNA levels through the regulation of both transcription and degradation. Organisms must degrade not only aberrantly folded or misprocessed RNAs, but also functional RNA transcripts that are no longer needed by the cell. In order to distinguish among and degrade only the appropriate RNA transcripts, cells have developed multiple RNA degradation processes and complexes. RNA degradation can occur by digestion inwards from the ends, using 5' to 3' and 3' to 5' exonucleases, or by digestion at internal sites using endonucleases. RNA degradation often, though not always, also involves deadenylation (for both 5' to 3' and 3' to 5' degradation) and decapping (for 5' to 3' degradation) of the transcripts. The range of RNA degradation machinery includes both nuclear and cytoplasmic components.
RNA degradation typically begins with the shortening of the long poly-A tail of mRNA transcripts. Although some RNA degradation pathways can apparently act on polyadenylated transcripts, e.g. nonstop decay (NSD), nonsense-mediated decay (NMD), and endonucleolytic cleavage (RNAi), the majority of 5' to 3' and 3' to 5' exonucleolytic activities require the prior removal of the poly-A tail. Two proteins, Ccr4p and/or Caf1p, have been shown to act as the catalytic core of the deadenylation machinery in all eukaryotic cells examined to date [1–3]. 5' to 3' degradation depends on the prior removal of the 5' cap structure followed by subsequent 5' to 3' exonucleolytic cleavage. In most cells this is performed by the Dcp1p/Dcp2p holoenzyme with the involvement of a wide and diverse array of additional protein machinery [4–6].
The exosome, a 3' to 5' exonuclease complex, is one of the important RNA degradation complexes and can be used as a means of classifying the different machinery into two groups: the exosome-dependent and the exosome-independent pathways. It is this convention that we use here to describe the RNA degradation machinery in Giardia lamblia and other parasitic protists. Exosomes have clear homologs in all three domains of life. Bacteria, archaea, and eukaryotes possess functionally analogous core 3' to 5' RNA degradation complexes in the bacterial polynucleotide phosphorylase (PNPase), the archaeal exosome, and the eukaryotic exosome, respectively. The similarity in the structure of all three mRNA degradation complexes is striking and suggests that the highly conserved structures are necessary for mRNA degradation and have been maintained throughout evolutionary history.
The bacterial PNPase exists as a homotrimer, in which each monomer possesses two tandem RNase PH domains in addition to single S1 and KH domains . RNase PH domains have exonucleolytic activity, although only one of the two domains in each monomer is thought to be active, while the S1 and KH domains have RNA binding capacity . The archaeal exosome ring is composed of repeating Rrp41/42 heterodimers arranged into a hexamer with three total copies of the stabilizing proteins Rrp4 and Csl4 acting as caps to the complex. The Rrp41 and Rrp42 subunits possess RNase PH domains, but Rrp41 is the only exonucleolytic component of the complex, again resulting in three active sites per complex [7, 8]. Rrp4 and Csl4 both possess S1 domains and bind RNA.
In eukaryotes, the core exosome also exists as a ring structure made of a heterohexamer of proteins with RNase PH domains (the three Rrp 41-like proteins are Rrps41, 46 and Mtr3, and the three Rrp 42-like proteins are Rrps42, 43, and 45) with a trio of additional RNA-binding proteins which contain S1 domains (Rrps 4, 40, and Csl4) that broadly act as the entry to the pore of the exosome and in eukaryotic exosomes further act to stabilize the hexameric ring [9, 10]. It is believed that, through gene duplication either early in the eukaryotic lineage or prior to the divergence of eukaryotes, rrp41 gave rise to both rrp46 and mtr3, while rrp42 gave rise to rrp43 and rrp45 . The ring and stabilizing proteins are commonly associated with Rrp6 and Rrp44, both of which possess nucleolytic activity [9, 11]. The core proteins display homology to archaeal exosome and bacterial PNPase proteins, whereas Rrp6 and Rrp44 display homology to bacterial RNases [12–14]. In some eukaryotes, the RNase PH domain of Rrp41 provides exonucleolytic activity, whereas in other species the activity is dependent upon Rrp6 and Rrp44 [9, 11, 15].
Although the exosome is an important complex involved in RNA degradation in eukaryotes, additional complexes also play a role in RNA degradation either through exosome-dependent or exosome-independent processes. Exosome-dependent complexes act mainly by preparing RNA substrates for degradation by the exosome, whereas exosome-independent complexes possess nucleolytic activity of their own. These additional complexes impart specificity to its function so that RNA is not degraded prematurely, and only a subset of RNA is targeted at any one time.
The exosome-dependent machinery includes the TRAMP complex, Pumilio (Puf) proteins, Nonsense mediated decay (NMD), Nonstop decay (NSD), and No-go decay (NGD) complexes. In the nucleus, the exosome can be found to be associated with the TRAMP complex, which aids in the degradation, maturation, and removal of secondary structures of RNA molecules through the post-transcriptional addition of a poly-A tail by TRAMP proteins Trf4/5 or via the helicase activity of TRAMP protein Mtr4, respectively [16, 17]. In the cytoplasm a subset of Puf proteins bind mRNAs via sequence-specific elements in the 3' untranslated regions (UTRs) and recruit the deadenylation machinery [18, 19]. Also in the cytoplasm, the NMD, NSD, and NGD pathways act as mRNA quality control and are activated in response to mRNAs containing premature termination codons (PTCs), no stop codon, or secondary structures such as stem loops, respectively. The NMD complex may possess endonucleolytic activity, but requires the exosome for complete degradation of RNAs.
The exosome-independent complexes are the RNAi machinery and the Ccr4-Not complex. RNAi acts to silence gene expression through endonucleolytic degradation of targeted mRNA transcripts or translation inhibition. RNAi machinery has been identified in a variety of eukaryotic organisms, from single-celled organisms to metazoans but is not ubiquitously present. Two components of the Ccr4-Not complex, which is conserved from S. cerevisiae to humans , have roles in mRNA deadenylation; Ccr4 and Caf1 deadenylate mRNA transcripts, although optimal degradation for many transcripts still requires the exosome .
The parasitic protists regulate gene expression through many different mechanisms, both transcriptional and post-transcriptional. Yet, while we understand much about transcriptional regulation, the study of mRNA degradation machinery in the parasitic protists is still in its early stages. This is perhaps especially surprising because RNA degradation is likely to play an unusually prominent role in organisms that exhibit diminished regulation of gene expression at the transcriptional level, as is known to be the case for several parasitic protists. For example, Trypanosoma brucei transcribes its genes polycistronically, implying that mRNA processing and degradation are its primary means of regulating gene expression [22–24]. And Giardia lamblia transcribes an abundance of full-length sterile antisense transcripts that are capped and polyadenylated , suggesting a role for mRNA degradation to eliminate these aberrant transcripts.
In this paper, we discuss our efforts to identify the mRNA degradation machinery in Giardia lamblia using in silico approaches. We additionally included several other parasitic protists (Entamoeba histolytica, Trichomonas vaginalis, Trypansoma brucei, and Plasmodium falciparum) in our analyses of the core and peripheral exosome components for comparison, building on the work of previous researchers in this field [1, 23, 24, 26–33]. We focused especially on Giardia given its evolutionary divergence [34, 35], severely reduced repertoire of transcriptional machinery [34, 36], and unusual patterns of gene expression [37, 38]. We identified an extensive collection of genes coding for proteins with significant sequence similarity to proteins that participate in RNA degradation pathways in other eukaryotes. Pathways such as the RNAi [39–41] and nonsense mediated decay pathways [28, 42] previously have been identified in Giardia. However, these comparisons also revealed that a substantial number of protein constituents of mRNA degradation complexes in other eukaryotes are either absent or sufficiently divergent to thwart detection by similarity searches in these parasitic protists. We use this new knowledge to consider which protein components may comprise the most reduced core exosome structure in eukaryotes and to postulate explanations for observed patterns of mRNA transcripts in Giardia.