Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing

Dicer proteins are central to the different mechanisms involving RNA interference. Plants have evolved multiple DICER‐LIKE (DCL) copies, thus enabling functional diversification. In Arabidopsis, DCL2 and DCL4 process double‐stranded RNA into 22 and 21 nucleotide small interfering (si)RNAs, respectively, and have overlapping functions with regards to virus and transgene silencing. Nonetheless, some studies have reported that dcl2 or dcl4 single mutations are sometimes sufficient to hinder silencing. To better dissect the role of DCL2 and DCL4, we analyzed silencing kinetics and efficiencies using different transgenic systems in single and double mutant backgrounds. The results indicate that DCL2 stimulates transitivity and secondary siRNA production through DCL4 while being sufficient for silencing on its own. Notably, silencing of 35S‐driven transgenes functions more efficiently in dcl4 mutants, indicating that DCL4 mostly obscures DCL2 in wild‐type plants. Nonetheless, in a dcl4 mutant compromised in phloem‐originating silencing, ectopically expressed DCL2 allows restoration of silencing, suggesting that DCL2 is not, or poorly, expressed in phloem. Remarkably, this ectopic DCL2 contribution to phloem‐originating silencing is dependent on the activity of RNA‐DEPENDENT RNA POLYMERASE6. These results indicate that, despite differences in the silencing activity of their small RNA products, DCL2 and DCL4 mostly act redundantly yet hierarchically when present simultaneously.     

植物小RNA的研究进展

在植物中发现大量内源性的小RNA,它们与真核生物中的内源性的微RNA和外源性的干扰小RNA有类似的性质和功能。本对植物中小RNA分子的分布、作用机制、功能以及信号传导等方面作一概述。     

Potential coexistence of both bacterial and eukaryotic small RNA biogenesis and functional related protein homologs in Archaea

RNA silencing plays crucial roles in both bacteria and eukaryotes, yet its machinery appears to differ in these two kingdoms. A couple of Argonaute protein homologs have been reported in some archaeal species in recent years. As Argonaute protein is the key component of eukaryotic RNA silencing pathways, such findings suggested the possibility of existence of eukaryotic RNA silencing like pathways in Archaea, which present the life forms between prokaryotes and eukaryotes. To further explore such hypothesis, we systematically screened 71 fully sequenced archaeal genomes, and identified some proteins containing homologous regions to the functional domains of eukaryotie RNA silencing pathway key proteins. The phylogenetic relationships of these proteins were analyzed. The conserved time-tional amino acids between archaeal and eukaryotic Piwi domains suggested their functional similarity. Our results provide new clues to the evolution of RNA silencing pathways.     

Development of reduced-fat mayonnaise using 4αGTase-modified rice starch and xanthan gum

In this study a disproportionating enzyme, 4-α-glucanotransferase (4αGTase), was used to modify the structural properties of rice starch to produce a suitable fat substitute in reduced-fat (RF) mayonnaise. The mayonnaise fat was partially substituted with the 4αGTase-treated starch paste at levels up to 50% in combination with xanthan gum and the physical and rheological properties of the modified RF mayonnaise samples were investigated. All mayonnaises prepared in this study exhibited shear thinning behavior and yield stress. Viscoelastic properties of mayonnaise were characterized using dynamic oscillatory shear test and it was observed that mayonnaises exhibited weak gel-like properties. The magnitude of elastic and loss moduli was also affected by 4αGTase-treated starch concentration and presence of xanthan gum. In relation to microstructure, RF mayonnaise prepared with 3.8 or 5.6 wt% of 4αGTase-treated starch and xanthan gum showed smaller droplets. The use of 5.6 wt% of 4αGTase-treated starch and 0.1 wt% of xanthan gum produced a RF mayonnaise with similar rheological properties and appearances as FF mayonnaise with gum. This study demonstrated a high feasibility for using 4αGTase-treated rice starch as a viable fat replacer in mayonnaise.     

Regulation of small RNA stability: methylation and beyond

As central components of RNA silencing, small RNAs play diverse and important roles in many biological processes in eukaryotes. Aberrant reduction or elevation in the levels of small RNAs is associated with many developmental and physiological defects. The in vivo levels of small RNAs are precisely regulated through modulating the rates of their biogenesis and turnover. 2′-O-methylation on the 3′ terminal ribose is a major mechanism that increases the stability of small RNAs. The small RNA methyltransferase HUA ENHANCER1 (HEN1) and its homologs methylate microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs (piRNAs) in animals, and siRNAs in Drosophila. 3′ nucleotide addition, especially uridylation, and 3′-5′ exonucleolytic degradation are major mechanisms that turnover small RNAs. Other mechanisms impacting small RNA stability include complementary RNAs, cis-elements in small RNA sequences and RNA-binding proteins. Investigations are ongoing to further understand how small RNA stability impacts their accumulation in vivo in order to improve the utilization of RNA silencing in biotechnology and therapeutic applications.     

Recovery of dicer-like 1-late flowering phenotype by miR172 expressed by the noncanonical DCL4-dependent biogenesis pathway

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.     

Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.     

MTR4, a putative RNA helicase and exosome co-factor, is required for proper rRNA biogenesis and development in Arabidopsis thaliana

The exosome is a conserved protein complex that is responsible for essential 3'→5' RNA degradation in both the nucleus and the cytosol. It is composed of a nine-subunit core complex to which co-factors confer both RNA substrate recognition and ribonucleolytic activities. Very few exosome co-factors have been identified in plants. Here, we have characterized a putative RNA helicase, AtMTR4, that is involved in the degradation of several nucleolar exosome substrates in Arabidopsis thaliana. We show that AtMTR4, rather than its closely related protein HEN2, is required for proper rRNA biogenesis in Arabidopsis. AtMTR4 is mostly localized in the nucleolus, a subcellular compartmentalization that is shared with another exosome co-factor, RRP6L2. AtMTR4 and RRP6L2 cooperate in several steps of rRNA maturation and surveillance, such as processing the 5.8S rRNA and removal of rRNA maturation by-products. Interestingly, degradation of the Arabidopsis 5' external transcribed spacer (5' ETS) requires cooperation of both the 5'→3' and 3'→5' exoribonucleolytic pathways. Accumulating AtMTR4 targets give rise to illegitimate small RNAs; however, these do not affect rRNA metabolism or contribute to the phenotype of mtr4 mutants. Plants lacking AtMTR4 are viable but show several developmental defects, including aberrant vein patterning and pointed first leaves. The mtr4 phenotype resembles that of several ribosomal protein and nucleolin mutants, and may be explained by delayed ribosome biogenesis, as we observed a reduced rate of rRNA accumulation in mtr4 mutants. Taken together, these data link AtMTR4 with rRNA biogenesis and development in Arabidopsis.     

Are γδ T cells important for the elimination of virus-infected cells?

Rhesus monkeys (Macaca mulatta) gamma delta T cells were identified using a monoclonal antibody. The relative representation of gamma delta T lymphocytes in the peripheral blood, lymph nodes, and spleen resembles that of Homo sapiens. The analysis of function and specificity revealed further significant similarities between the simian and human gamma delta T-cell systems. Since both human and monkey gamma delta T lymphocytes can effectively lyse cells infected with immunodeficiency viruses, it is possible that the primate gamma delta T-cell systems contribute to antiviral immunosurveillance.     

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.     

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

Interferon-γ activates the cleavage of double-stranded RNA by bovine seminal ribonuclease

Bovine seminal ribonuclease (BS-RNase), a dimeric homologue of RNase A, cleaves both single- and double-stranded RNA and inhibits the growth of tumor cells. Its catalytic activity against double-stranded RNA, either homopolymeric ([³H]polyA/polyU) or mixed sequence, is enhanced by bovine or human recombinant interferon-γ (IFN-γ). Activation is seen with as little as 4–10 interferon units per assay. Enhancing the degradation of double-stranded RNA, an intermediate in the growth cycle of many viruses, could contribute to IFN-γ's ability to control cell growth and induce an antiviral state.     

Identification and characterization of small RNAs involved in RNA silencing

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.     

Induction of mouse β integrin expression following transfection with human α4 chain

We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.