Nuclear Domains Enriched in RNA 3′-processing Factors Associate with Coiled Bodies and Histone Genes in a Cell Cycle–dependent Manner

Nuclear domains, called cleavage bodies, are enriched in the RNA 3′-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle–dependent expression of histone genes.

RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery

Multiple members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in A-to-I RNA editing. It has been speculated that they may form a large multicomponent protein complex. Possible candidates for such complexes are large nuclear ribonucleoprotein (lnRNP) particles. The lnRNP particles consist mainly of four spliceosomal subunits that assemble together with the pre-mRNA to form a large particle and thus are viewed as the naturally assembled pre-mRNA processing machinery. Here we investigated the presence of ADARs in lnRNP particles by Western blot analysis using anti-ADAR antibodies and by indirect immunoprecipitation. Both ADAR1 and ADAR2 were found associated with the spliceosomal components Sm and SR proteins within the lnRNP particles. The two ADARs, associated with lnRNP particles, were enzymatically active in site-selective A-to-I RNA editing. We demonstrate the association of ADAR RNA editing enzymes with physiological supramolecular complexes, the lnRNP particles.

The tRNA processing enzyme RNase T is essential for maturation of 5S RNA.

The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival.

Developmentally and transgene regulated nuclear processing of primary transcripts of chalcone synthase A in petunia

The introduction of chalcone synthase A transgenes into petunia plants can result in degradation of chalcone synthase A RNAs and loss of chalcone synthase, a process called cosuppression or post-transcriptional gene silencing. Here we show that the RNA degradation is associated with changes in premRNA processing, i.e. loss of tissue specificity in transcript cleavage patterns, accumulation of unspliced molecules, and use of template-specific secondary poly(A) sites. These changes can also be observed at a lower level in leaves but not flowers of nontransgenic petunias. Based on this, a model is presented of how transgenes may disturb the carefully evolved, developmentally controlled post-transcriptional regulation of chalcone synthase gene expression by influencing the survival rate of the endogenous and their own mRNA.

Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination

Pcf11p, an essential subunit of the yeast cleavage factor IA, is required for pre‐mRNA 3′ end processing, binds to the C‐terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) and is involved in transcription termination. We show that the conserved CTD interaction domain (CID) of Pcf11p is essential for cell viability. Interestingly, the CTD binding and 3′ end processing activities of Pcf11p can be functionally uncoupled from each other and provided by distinct Pcf11p fragments in trans. Impaired CTD binding did not affect the 3′ end processing activity of Pcf11p and a deficiency of Pcf11p in 3′ end processing did not prevent CTD binding. Transcriptional run‐on analysis with the CYC1 gene revealed that loss of cleavage activity did not correlate with a defect in transcription termination, whereas loss of CTD binding did. We conclude that Pcf11p is a bifunctional protein and that transcript cleavage is not an obligatory step prior to RNAP II termination.

Folates in rye: Determination and enhancement by food processing

The average daily intake of folate, one of the B vitamins, falls below recommendations among the Finnish population. Bread and cereals are the main sources of folate, rye being the most significant single source. Processing is a prerequisite for the consumption of whole grain rye; however, little is known about the effect of processing on folates. Moreover, data on the bioavailability of endogenous cereal folates are scarce. The aim of this study was to examine the variation in as well as the effect of fermentation, germination, and thermal processes on folate contents in rye. Bioavailability of endogenous rye folates was investigated in a four-week human intervention study. One of the objectives throughout the work was to optimise and evaluate analytical methods for determining folate contents in cereals. Affinity chromatographic purification followed by high-performance liquid chromatography (HPLC) was a suitable method for analysing cereal products for folate vitamers, and microbiological assay with Lactobacillus rhamnosus reliably quantified the total folate. However, HPLC gave approximately 30% lower results than the microbiological assay. The folate content of rye was high and could be further increased by targeted processing. The vitamer distribution of whole grain rye was characterised by a large proportion of formylated vitamers followed by 5-methyltetrahydrofolate. In sourdough fermentation of rye, the studied yeasts synthesized and lactic acid bacteria mainly depleted folate. Two endogenous bacteria isolated from rye flour were found to produce folate during fermentation. Inclusion of baker s yeast in sourdough fermentation raised the folate level so that the bread could contain more folate than the flour it was made of. Germination markedly increased the folate content of rye, with particularly high folate concentrations in hypocotylar roots. Thermal treatments caused significant folate losses but the preceding germination compensated well for the losses. In the bioavailability study, moderate amounts of endogenous folates in the form of different rye products and orange juice incorporated in the diet improved the folate status among healthy adults. Endogenous folates from rye and orange juice showed similar bioavailability to folic acid from fortified white bread. In brief, it was shown that the folate content of rye can be enhanced manifold by optimising and combining food processing techniques. This offers some practical means to increase the daily intake of folate in a bioavailable form.

Mechanisms for alphavirus nonstructural polyprotein processing

Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.

Innate Immune Recognition of RNA-virus infection in human macrophages

Innate immunity and host defence are rapidly evoked by structurally invariant molecular motifs common to microbial world, called pathogen associated molecular patterns (PAMPs). In addition to PAMPs, endogenous molecules released in response to inflammation and tissue damage, danger associated molecular patterns (DAMPs), are required for eliciting the response. The most important PAMPs of viruses are viral nucleic acids, their genome or its replication intermediates, whereas the identity and characteristics of virus infection-induced DAMPs are poorly defined. PAMPs and DAMPs engage a limited set of germ-line encoded pattern recognition receptors (PRRs) in immune and non-immune cells. Membrane-bound Toll-like receptors (TLRs), cytoplasmic retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptor (NLRs) are important PRRs involved in the recognition of the molecular signatures of viral infection, such as double-stranded ribonucleic acids (dsRNAs). Engagement of PRRs results in local and systemic innate immune responses which, when activated against viruses, evoke secretion of antiviral and pro-inflammatory cytokines, and programmed cell death i.e., apoptosis of the virus-infected cell. Macrophages are the central effector cells of innate immunity. They produce significant amounts of antiviral cytokines, called interferons (IFNs), and pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-18. IL-1β and IL-18 are synthesized as inactive precursors, pro-IL-1β and pro-IL-18, that are processed by caspase-1 in a cytoplasmic multiprotein complex, called the inflammasome. After processing, these cytokines are biologically active and will be secreted. The signals and secretory routes that activate inflammasomes and the secretion of IL-1β and IL-18 during virus infections are poorly characterized. The main goal of this thesis was to characterize influenza A virus-induced innate immune responses and host-virus interactions in human primary macrophages during an infection. Methodologically, various techniques of cellular and molecular biology, as well as proteomic tools combined with bioinformatics, were utilized. Overall, the thesis provides interesting insights into inflammatory and antiviral innate immune responses, and has characterized host-virus interactions during influenza A virus-infection in human primary macrophages.

Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 3 ` and 5 ` End Repair and Role of Polyprotein Processing in Viral Replication

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.

Proteolytic processing of the nonstructural proteins of dengue 2 virus

The genomes of many positive stranded RNA viruses and of all retroviruses are translated as large polyproteins which are proteolytically processed by cellular and viral proteases. Viral proteases are structurally related to two families of cellular proteases, the pepsin-like and trypsin-like proteases. This thesis describes the proteolytic processing of several nonstructural proteins of dengue 2 virus, a representative member of the Flaviviridae, and describes methods for transcribing full-length genomic RNA of dengue 2 virus. Chapter 1 describes the in vitro processing of the nonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a system that allows identification of residues within the protease that are directly or indirectly involved with substrate recognition. Chapter 3 describes methods to produce genome length dengue 2 RNA from cDNA templates.

The nonstructural protein NS3 is structurally related to viral trypsinlike proteases from the alpha-, picorna-, poty-, and pestiviruses. The hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins was tested using an efficient in vitro expression system and antisera specific for the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed using T7 RNA polymerase and the RNA translated in reticulocyte lysates. Proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain necessary and sufficient for correct cleavage to the first 184 amino acids of NS3. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.

Biochemical and genetic experiments using viral proteinases have defined the sequence requirements for cleavage site recognition, but have not identified residues within proteinases that interact with substrates. A biochemical assay was developed that could identify residues which were important for substrate recognition. Chimeric proteases between yellow fever and dengue 2 were constructed that allowed mapping of regions involved in substrate recognition, and site directed mutagenesis was used to modulate processing efficiency.

Expression in vitro revealed that the dengue protease domain efficiently processes the yellow fever polyprotein between NS2A and NS2B and between NS2B and NS3, but that the reciprocal construct is inactive. The dengue protease processes yellow fever cleavage sites more efficiently than dengue cleavage sites, suggesting that suboptimal cleavage efficiency may be used to increase levels of processing intermediates in vivo. By mutagenizing the putative substrate binding pocket it was possible to change the substrate specificity of the yellow fever protease; changing a minimum of three amino acids in the yellow fever protease enabled it to recognize dengue cleavage sites. This system allows identification of residues which are directly or indirectly involved with enzyme-substrate interaction, does not require a crystal structure, and can define the substrate preferences of individual members of a viral proteinase family.

Full-length cDNA clones, from which infectious RNA can be transcribed, have been developed for a number of positive strand RNA viruses, including the flavivirus type virus, yellow fever. The technology necessary to transcribe genomic RNA of dengue 2 virus was developed in order to better understand the molecular biology of the dengue subgroup. A 5' structural region clone was engineered to transcribe authentic dengue RNA that contains an additional 1 or 2 residues at the 5' end. A 3' nonstructural region clone was engineered to allow production of run off transcripts, and to allow directional ligation with the 5' structural region clone. In vitro ligation and transcription produces full-length genomic RNA which is noninfectious when transfected into mammalian tissue culture cells. Alternative methods for constructing cDNA clones and recovering live dengue virus are discussed.

An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana.

ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.

Role of RNA polymerase IV in plant small RNA metabolism.

In addition to the three RNA polymerases (RNAP I-III) shared by all eukaryotic organisms, plant genomes encode a fourth RNAP (RNAP IV) that appears to be specialized in the production of siRNAs. Available data support a model in which dsRNAs are generated by RNAP IV and RNA-dependent RNAP 2 (RDR2) and processed by DICER (DCL) enzymes into 21- to 24-nt siRNAs, which are associated with different ARGONAUTE (AGO) proteins for transcriptional or posttranscriptional gene silencing. However, it is not yet clear what fraction of genomic siRNA production is RNAP IV-dependent, and to what extent these siRNAs are preferentially processed by certain DCL(s) or associated with specific AGOs for distinct downstream functions. To address these questions on a genome-wide scale, we sequenced approximately 335,000 siRNAs from wild-type and RNAP IV mutant Arabidopsis plants by using 454 technology. The results show that RNAP IV is required for the production of >90% of all siRNAs, which are faithfully produced from a discrete set of genomic loci. Comparisons of these siRNAs with those accumulated in rdr2 and dcl2 dcl3 dcl4 and those associated with AGO1 and AGO4 provide important information regarding the processing, channeling, and functions of plant siRNAs. We also describe a class of RNAP IV-independent siRNAs produced from endogenous single-stranded hairpin RNA precursors.

New perspectives on neoplasia and the RNA world

Key tenets of modern biology are the central place of protein in cell regulation and the flow of genetic information from DNA to RNA to protein. However, it is becoming increasingly apparent that genomes are much more complex than hitherto thought with remarkably complex regulatory systems. The notion that the fraction of the genome involved in coding protein is all that matters is increasingly being questioned as the roles of non-coding RNA (ncRNA) in cellular systems becomes recognised. The RNA world, including microRNA (miRNA), small inhibitory RNA (siRNA) and other RNA species, are now recognised as being crucial for the regulation of chromatin structure, gene expression, mRNA processing and splicing, mRNA stability and translational control. Furthermore such ncRNA systems may be perturbed in disease states and most notably in neoplasia, including in haematological malignancies. Here the burgeoning evidence for a role of miRNA in neoplasia is reviewed and the importance of understanding the RNA world emphasised. Copyright (c) 2005 John Wiley & Sons, Ltd.

Targeted 3' processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing.

Noncoding RNA is emerging as an important regulator of gene expression in many organisms. We are characterizing RNA-mediated chromatin silencing of the Arabidopsis major floral repressor gene, FLC. Through suppressor mutagenesis, we identify a requirement for CstF64 and CstF77, two conserved RNA 3'-end-processing factors, in FLC silencing. However, FLC sense transcript 3' processing is not affected in the mutants. Instead, CstF64 and CstF77 are required for 3' processing of FLC antisense transcripts. A specific RNA-binding protein directs their activity to a proximal antisense polyadenylation site. This targeted processing triggers localized histone demethylase activity and results in reduced FLC sense transcription. Targeted 3' processing of antisense transcripts may be a common mechanism triggering transcriptional silencing of the corresponding sense gene.

Dysregulated ADAM10-Mediated Processing of APP during a Critical Time Window Leads to Synaptic Deficits in Fragile X Syndrome.

The Fragile X mental retardation protein (FMRP) regulates neuronal RNA metabolism, and its absence or mutations leads to the Fragile X syndrome (FXS). The β-amyloid precursor protein (APP) is involved in Alzheimer's disease, plays a role in synapse formation, and is upregulated in intellectual disabilities. Here, we show that during mouse synaptogenesis and in human FXS fibroblasts, a dual dysregulation of APP and the α-secretase ADAM10 leads to the production of an excess of soluble APPα (sAPPα). In FXS, sAPPα signals through the metabotropic receptor that, activating the MAP kinase pathway, leads to synaptic and behavioral deficits. Modulation of ADAM10 activity in FXS reduces sAPPα levels, restoring translational control, synaptic morphology, and behavioral plasticity. Thus, proper control of ADAM10-mediated APP processing during a specific developmental postnatal stage is crucial for healthy spine formation and function(s). Downregulation of ADAM10 activity at synapses may be an effective strategy for ameliorating FXS phenotypes.