The exosome complex plays a central and essential role in RNA metabolism. However, comprehensive studies of exosome substrates and functional analyses of its subunits are lacking. Here, we demonstrate that as opposed to yeast and metazoans the plant exosome core possesses an unanticipated functional plasticity and present a genome-wide atlas of Arabidopsis exosome targets. Additionally, our study provides evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants, reveals unexpected aspects of stable structural RNA metabolism, and uncovers numerous novel exosome substrates. These include a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described and belong to a layer of the transcriptome that can only be visualized upon inhibition of exosome activity. These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes.

Eukaryotic genomes encode thousands of long noncoding RNAs (lncRNAs), which play important roles in essential biological processes. Although lncRNAs function in the nuclear and cytoplasmic compartments, most of them occur in the nucleus, often in association with chromatin. Indeed, many lncRNAs have emerged as key regulators of gene expression and genome stability. Emerging evidence also suggests that lncRNAs may contribute to the organization of nuclear domains. This review briefly summarizes the major types of eukaryotic lncRNAs and provides examples of their mechanisms of action, with focus on plant lncRNAs, mainly in Arabidopsis thaliana, and describes current advances in our understanding of the mechanisms of lncRNA action and the roles of lncRNAs in RNA-dependent DNA methylation and in the regulation of flowering time.

As part of our genetic analysis of mRNA decay in Escherichia coli K-12, we Consequently, we set out to examine additional genes that might be involved. The identification of pcnB, the structural gene for poly(A) polymerase I (PAP I) (5), has allowed us to determine whether polyadenylylation affects mRNA decay in E. coli the same way it does in eukaryotes (6). For many eukaryotic mRNAs, the degradation of poly(A) tails initiates mRNA decay (7-9). If it does so in E. coli, then, presumably, mRNA stability would increase in the absence of PAP I.To test whether the degradation of poly(A) tails initiates mRNA decay in E. coli, a series of isogenic strains containing mutations in PAP I (10), RNase E(mne) (1), PNPase (pnp) (1), and RNase II (mb) (1) were constructed. Northern analysis of the trxA, ompA, and lpp mRNAs showed that when polyadenylylation was almost completely eliminated, their half-lives always increased significantly and their decay patterns changed. We also determined the number and size of poly(A) tails in the total bacterial RNA population. In wild-type E. coli, poly(A) tails ranged from 10 to >50 nt. Where there was no PNPase and there was reduced RNase II activity, both the number and the size of the tails increased significantly. More than 90% of the poly(A) tails were absent in ApcnB mutants. We discuss the implications of these results below.MATERIALS AND METHODS Bacterial Strains. All strains were derived from E. coli MG1693 (thyA715), provided by B. Bachmann (E. coli Genetic Stock Center, Yale University). SK5704 (pnp-7 mb-500 me-i thyA 715) (1) was constructed by phage P1-mediated transduction (11). SK7988 (ApcnB thyA715) and SK8901 (lApcnBpnp-7 mb-500 me-I thyA 715) were derived from MG1693 and SK5704, respectively. A kanamycin-resistance (KmR) determinant in the ApcnB gene (10) was used as a selectable transduction marker. SK7988 and SK8901 each grew more slowly than their pcnB+ parents. The absence of poly(A) polymerase in SK8901 did not affect the conditional lethality associated with inactivating RNase II, RNase E, and PNPase.RNA Isolation. Seven-milliliter samples were mixed with an equal volume of crushed frozen TM buffer (10 mM Tris, pH 7.2/5 mM MgCl2) containing 20 mM NaN3 and chloramphenicol at 0.4 mg/ml. The cells were then centrifuged and the pellet was suspended in 0.34 ml of TM buffer with lysozyme at 0.3 mg/ml and DNase I at 32 units/ml. After three freezethaw cycles, one-sixth volume of 20 mM acetic acid was added, followed by an equal volume of Catrimox-14 (Iowa Biotechnology, Oakdale, IA).The resulting precipitate of protein, DNA, and RNA was spun in a microcentrifuge at medium speed to form a soft pellet. That pellet was completely suspended in 1 ml of 2 M LiCl in 35% ethanol. In this solution protein and DNA dissolve but RNA does not. The resulting RNA precipitate was centrifuged at high speed for 5 min to form a pellet. It was then suspended in 2 M LiCl in distilled water and centrifuged again. The pellet was washed with 70% ethanol and resuspended in distilled w...

Multiple studies indicate that mRNA processing defects cause mRNAs to accumulate in discrete nuclear foci or dots, in mammalian cells as well as yeast. To investigate this phenomenon, we have studied a series of GAL reporter constructs integrated into the yeast genome adjacent to an array of TetR-GFP-bound TetO sites. mRNA within dots is predominantly post-transcriptional, and dots are adjacent to but distinct from their transcription site. These reporter genes also localize to the nuclear periphery upon gene induction, like their endogenous GAL counterparts. Surprisingly, this peripheral localization persists long after transcriptional shutoff, and there is a comparable persistence of the RNA in the dots. Moreover, dot disappearance and gene delocalization from the nuclear periphery occur with similar kinetics after transcriptional shutoff. Both kinetics depend in turn on reporter gene 3 0 -end formation signals. Our experiments indicate that gene association with the nuclear periphery does not require ongoing transcription and suggest that the mRNPs within dots may make a major contribution to the gene-nuclear periphery tether.

Proteins bound to the poly(A) tail of mRNA transcripts, called poly(A)-binding proteins (Pabs), play critical roles in regulating RNA stability, translation, and nuclear export. Like many mRNA-binding proteins that modulate post-transcriptional processing events, assigning specific functions to Pabs is challenging because these processing events are tightly coupled to one another. To investigate the role that a novel class of zinc finger-containing Pabs plays in these coupled processes, we defined the mode of polyadenosine RNA recognition for the conserved Saccharomyces cerevisiae Nab2 protein and assessed in vivo consequences caused by disruption of RNA binding. The polyadenosine RNA recognition domain of Nab2 consists of three tandem Cys-Cys-Cys-His (CCCH) zinc fingers. Cells expressing mutant Nab2 proteins with decreased binding to polyadenosine RNA show growth defects as well as defects in poly(A) tail length but do not accumulate poly(A) RNA in the nucleus. We also demonstrate genetic interactions between mutant nab2 alleles and mutant alleles of the mRNA 3′-end processing machinery. Together, these data provide strong evidence that Nab2 binding to RNA is critical for proper control of poly(A) tail length.

The eukaryotic genomes are pervasively transcribed. In addition to protein-coding RNAs, thousands of long noncoding RNAs (lncRNAs) modulate key molecular and biological processes. Most lncRNAs are found in the nucleus and associate with chromatin, but lncRNAs can function in both nuclear and cytoplasmic compartments. Emerging work has found that many lncRNAs regulate gene expression and can affect genome stability and nuclear domain organization both in plant and in the animal kingdom. Here, we describe the major plant lncRNAs and how they act, with a focus on research in Arabidopsis thaliana and our emerging understanding of lncRNA functions in serving as molecular sponges and decoys, functioning in regulation of transcription and silencing, particularly in RNA-directed DNA methylation, and in epigenetic regulation of flowering time.

Deadenylation of mRNA is often the first and rate-limiting step in mRNA decay. PARN, a poly(A)-specific 3' --> 5' ribonuclease which is conserved in many eukaryotes, has been proposed to be primarily responsible for such a reaction, yet the importance of the PARN function at the whole-organism level has not been demonstrated in any species. Here, we show that mRNA deadenylation by PARN is essential for viability in higher plants (Arabidopsis thaliana). Yet, this essential requirement for the PARN function is not universal across the phylogenetic spectrum, because PARN is dispensable in Fungi (Schizosaccharomyces pombe), and can be at least severely downregulated without any obvious consequences in Metazoa (Caenorhabditis elegans). Development of the Arabidopsis embryos lacking PARN (AtPARN), as well as of those expressing an enzymatically inactive protein, was markedly retarded, and ultimately culminated in an arrest at the bent-cotyledon stage. Importantly, only some, rather than all, embryo-specific transcripts were hyperadenylated in the mutant embryos, suggesting that preferential deadenylation of a specific select subset of mRNAs, rather than a general deadenylation of the whole mRNA population, by AtPARN is indispensable for embryogenesis in Arabidopsis. These findings indicate a unique, nonredundant role of AtPARN among the multiple plant deadenylases.

Eukaryotic 335 exonucleolytic activities are essential for a wide variety of reactions of RNA maturation and metabolism, including processing of rRNA, small nuclear RNA, and small nucleolar RNA, and mRNA decay. Two related but distinct forms of a complex containing 10 335 exonucleases, the exosome, are found in yeast nucleus and cytoplasm, respectively, and related complexes exist in human cells. Here we report on the characterization of the AtRrp41p, an Arabidopsis thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p). Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease activity and requires a single-stranded poly(A) tail on a substrate RNA as a "loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the 5.8 S rRNA processing and 335 mRNA degradation defects of the yeast ski6 -100 mutant. However, neither of these defects can explain the conditional lethal phenotype of the ski6 -100 strain. Importantly, AtRrp41p shares additional function(s) with the yeast Rrp41p which are essential for cell viability because it also rescues the rrp41 (ski6) null mutant. AtRrp41p is found predominantly in a high molecular mass complex in Arabidopsis and in yeast cells, and it interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting that it can participate in evolutionarily conserved interactions that could be essential for the integrity of the exosome complex.A large number of eukaryotic RNA species require 3Ј35Ј exonucleolytic activities either for processing from their respective precursor forms or for their turnover. In yeast, a large, ϳ300 -400 kDa complex, the exosome, mediates many of these reactions (2, 21; for review, see Ref. 26). Two major forms of the exosome, nuclear and cytoplasmic, have been identified, each containing at least 10 common "core" components, Rrp4p, Rrp40p-Rrp46p, Mtr3p, and Csl4p proteins. In addition, Rrp6p appears to be associated only with the nuclear form of the exosome. Furthermore, RNA helicases Dob1p and Ski2p are required for at least some activities of the nuclear and cytoplasmic forms of the exosome, respectively, although their physical associations with it have not been documented.Interestingly, all but one of the core exosome subunits either have been shown to be 3Ј35Ј exonucleases in vitro or were predicted to have such activity, based on sequence similarity to known exonucleases. Six of the exosome components (Rrp41p, Rrp42p, Rrp43p, Rrp45p, Rrp46p, and Mtr3p) are homologous to the Escherichia coli RNase PH, and the recombinant yeast Rrp41p has a phosphorolytic, processive 3Ј35Ј exonuclease activity (21). Rrp4p and Rrp40p are homologous to each other and, along with Csl4p, contain a predicted S1 RNA binding motif. Recombinant Rrp4p is a hydrolytic, distributive 3Ј35Ј exonuclease. Rrp44p is homologous to the E. coli RNase R, of the RNase II family, and exhibits a hydrolytic, processive mode of RNA degradation. Finally, Rrp6p is a member of the RNase D family, and the recombinant Rrp6p has a...