The Cid1 poly(U) polymerase

The Schizosaccharomyces pombe cytoplasmic protein Cid1 acts as a poly(U) polymerase (PUP). Polyadenylated actin mRNA, a target of this activity, is uridylated upon arrest in S phase and is likely to be one of many such Cid1 targets. This RNA uridylation pathway appears to be conserved, as Cid1 orthologs in Arabidopsis thaliana, Caenorhabditis elegans and humans display PUP activity either in vitro or in Xenopus laevis oocytes. Here, we review the literature on Cid1, other PUPs and uridylation, a conserved and previously under-appreciated mechanism of RNA regulation.     

Crystal structures of the Cid1 poly (U) polymerase reveal the mechanism for UTP selectivity

Polyuridylation is emerging as a ubiquitous post-translational modification with important roles in multiple aspects of RNA metabolism. These poly (U) tails are added by poly (U) polymerases with homology to poly (A) polymerases; nevertheless, the selection for UTP over ATP remains enigmatic. We report the structures of poly (U) polymerase Cid1 from Schizoscaccharomyces pombe alone and in complex with UTP, CTP, GTP and 3′-dATP. These structures reveal that each of the 4 nt can be accommodated at the active site; however, differences exist that suggest how the polymerase selects UTP over the other nucleotides. Furthermore, we find that Cid1 shares a number of common UTP recognition features with the kinetoplastid terminal uridyltransferases. Kinetic analysis of Cid1’s activity for its preferred substrates, UTP and ATP, reveal a clear preference for UTP over ATP. Ultimately, we show that a single histidine in the active site plays a pivotal role for poly (U) activity. Notably, this residue is typically replaced by an asparagine residue in Cid1-family poly (A) polymerases. By mutating this histidine to an asparagine residue in Cid1, we diminished Cid1’s activity for UTP addition and improved ATP incorporation, supporting that this residue is important for UTP selectivity.     

The slicing activity of miRNA-specific Argonautes is essential for the miRNA pathway in C. elegans

Among the set of Argonautes proteins encoded by metazoan genomes, some have conserved amino acids important for catalytic or slicing activity. The functional significance of these residues in microRNA (miRNA)-specific Argonautes in animals is still unclear since miRNAs do not induce site-specific cleavage of targeted messenger RNAs (mRNAs), unlike small interfering RNAs (siRNAs). Here, we report that miRNA-specific ALG-1 and ALG-2 Argonautes from Caenorhabditis elegans possess the slicing activity normally implicated in the siRNA-silencing pathway. We also find that ALG-1/2 can bind and use a Dicer-processed miRNA duplex to target mRNAs, suggesting an ability to displace RNA strands. Importantly, the slicing activity of ALG-1 or ALG-2 is essential for the miRNA pathway in vivo, as shown by the accumulation of truncated miRNA precursors and altered miRNA-induced silencing complex (miRISC) formation. Taken together, our data demonstrate that the slicing activity of Argonautes contributes to a new and unexpected step in the canonical miRNA pathway that occurs prior to miRISC loading in animals.     

Repertoire of Bovine miRNA and miRNA-Like Small Regulatory RNAs Expressed upon Viral Infection

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small RNAs characterized in mammals and to examine relationship between host miRNA expression and viral infection we used Illumina''s ultrahigh throughput sequencing approach. We sequenced three small RNA libraries prepared from cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with Bovine herpesvirus 1. We used a bioinformatics approach to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected 219 out of 356 known bovine miRNAs and 115 respective miRNA* sequences. In addition we identified five new bovine orthologs of known mammalian miRNAs and discovered 268 new cow miRNAs many of which are not identifiable in other mammalian genomes and thus might be specific to the ruminant lineage. In addition we found seven new bovine mirtron candidates. We also discovered 10 small nucleolar RNA (snoRNA) loci that give rise to small RNA with possible miRNA-like function. Results presented in this study extend our knowledge of the biology and evolution of small regulatory RNAs in mammals and illuminate mechanisms of small RNA biogenesis and function. New miRNA sequences and the original sequencing data have been submitted to miRNA repository (miRBase) and NCBI GEO archive respectively. We envisage that these resources will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals.     

A family of poly(U) polymerases

The GLD-2 family of poly(A) polymerases add successive AMP monomers to the 3' end of specific RNAs, forming a poly(A) tail. Here, we identify a new group of GLD-2-related nucleotidyl transferases from Arabidopsis, Schizosaccharomyces pombe, Caenorhabditis elegans, and humans. Like GLD-2, these enzymes are template independent and add nucleotides to the 3' end of an RNA substrate. However, these new enzymes, which we refer to as poly(U) polymerases, add poly(U) rather than poly(A) to their RNA substrates.     

HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2' OH of the 3' terminal nucleotide

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3′ terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21–24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2′ OH of the 3′ terminal nucleotide. Among various modifications that can occur on the ribose of the terminal nucleotide, such as 2′-deoxy, 3′-deoxy, 2′-O-methyl and 3′-O-methyl, only 2′-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specifically methylates miRNAs and siRNAs and implicate the importance of the 2′-O-methyl group in the biology of RNA silencing.     

Polyadenylation regulates the stability of Trypanosoma brucei mitochondrial RNAs

Polyadenylation of RNAs plays a critical role in modulating rates of RNA turnover and ultimately in controlling gene expression in all systems examined to date. In mitochondria, the precise mechanisms by which RNAs are degraded, including the role of polyadenylation, are not well understood. Our previous in organello pulse-chase experiments suggest that poly(A) tails stimulate degradation of mRNAs in the mitochondria of the protozoan parasite Trypanosoma brucei (Militello, K. T., and Read, L. K. (2000) Mol. Cell. Biol. 21, 731-742). In this report, we developed an in vitro assay to directly examine the effects of specific 3'-sequences on RNA degradation. We found that a salt-extracted mitochondrial membrane fraction preferentially degraded polyadenylated mitochondrially and non-mitochondrially encoded RNAs over their non-adenylated counterparts. A poly(A) tail as short as 5 nucleotides was sufficient to stimulate rapid degradation, although an in vivo tail length of 20 adenosines supported the most rapid decay. A poly(U) extension did not promote rapid RNA degradation, and RNA turnover was slowed by the addition of uridine residues to the poly(A) tail. To stimulate degradation, the poly(A) element must be located at the 3' terminus of the RNA. Finally, we demonstrate that degradation of polyadenylated RNAs occurs in the 3' to 5' direction through the action of a hydrolytic exonuclease. These experiments demonstrate that the poly(A) tail can act as a cis-acting element to facilitate degradation of T. brucei mitochondrial mRNAs.     

Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity

CCR4, an evolutionarily conserved member of the CCR4–NOT complex, is the main cytoplasmic deadenylase. It contains a C‐terminal nuclease domain with homology to the endonuclease‐exonuclease‐phosphatase (EEP) family of enzymes. We have determined the high‐resolution three‐dimensional structure of the nuclease domain of CNOT6L, a human homologue of CCR4, by X‐ray crystallography using the single‐wavelength anomalous dispersion method. This first structure of a deadenylase belonging to the EEP family adopts a complete α/β sandwich fold typical of hydrolases with highly conserved active site residues similar to APE1. The active site of CNOT6L should recognize the RNA substrate through its negatively charged surface. In vitro deadenylase assays confirm the critical active site residues and show that the nuclease domain of CNOT6L exhibits full Mg²⁺‐dependent deadenylase activity with strict poly(A) RNA substrate specificity. To understand the structural basis for poly(A) RNA substrate binding, crystal structures of the CNOT6L nuclease domain have also been determined in complex with AMP and poly(A) DNA. The resulting structures suggest a molecular deadenylase mechanism involving a pentacovalent phosphate transition.     

Artificial microRNA-mediated virus resistance in plants

RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.     

Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA

Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.     

Prp43/DHX15 exemplify RNA helicase multifunctionality in the gene expression network

Dynamic regulation of RNA folding and structure is critical for the biogenesis and function of RNAs and ribonucleoprotein (RNP) complexes. Through their nucleotide triphosphate-dependent remodelling functions, RNA helicases are key modulators of RNA/RNP structure. While some RNA helicases are dedicated to a specific target RNA, others are multifunctional and engage numerous substrate RNAs in different aspects of RNA metabolism. The discovery of such multitasking RNA helicases raises the intriguing question of how these enzymes can act on diverse RNAs but also maintain specificity for their particular targets within the RNA-dense cellular environment. Furthermore, the identification of RNA helicases that sit at the nexus between different aspects of RNA metabolism raises the possibility that they mediate cross-regulation of different cellular processes. Prominent and extensively characterized multifunctional DEAH/RHA-box RNA helicases are DHX15 and its Saccharomyces cerevisiae (yeast) homologue Prp43. Due to their central roles in key cellular processes, these enzymes have also served as prototypes for mechanistic studies elucidating the mode of action of this type of enzyme. Here, we summarize the current knowledge on the structure, regulation and cellular functions of Prp43/DHX15, and discuss the general concept and implications of RNA helicase multifunctionality.     

MicroRNA-independent roles of the RNase III enzymes Drosha and Dicer

The ribonuclease III enzymes Drosha and Dicer are renowned for their central roles in the biogenesis of microRNAs (miRNAs). For many years, this has overshadowed the true versatility and importance of these enzymes in the processing of other RNA substrates. For example, Drosha also recognizes and cleaves messenger RNAs (mRNAs), and potentially ribosomal RNA. The cleavage of mRNAs occurs via recognition of secondary stem-loop structures similar to miRNA precursors, and is an important mechanism of repressing gene expression, particularly in progenitor/stem cell populations. On the other hand, Dicer also has critical roles in genome regulation and surveillance. These include the production of endogenous small interfering RNAs from many sources, and the degradation of potentially harmful short interspersed element and viral RNAs. These findings have sparked a renewed interest in these enzymes, and their diverse functions in biology.