Functional consequences of subunit diversity in RNA polymerases II and V

SummaryMultisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V

The Arabidopsis Chromatin-Modifying Nuclear siRNA Pathway Involves a Nucleolar RNA Processing Center

SummaryIn Arabidopsis thaliana, small interfering RNAs (siRNAs) direct cytosine methylation at endogenous DNA repeats in a pathway involving two forms of nuclear RNA polymerase IV (Pol IVa and Pol IVb), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3), ARGONAUTE4 (AGO4), the chromatin remodeler DRD1, and the de novo cytosine methyltransferase DRM2. We show that RDR2, DCL3, AGO4, and NRPD1b (the largest subunit of Pol IVb) colocalize with siRNAs within the nucleolus. By contrast, Pol IVa and DRD1 are external to the nucleolus and colocalize with endogenous repeat loci. Mutation-induced loss of pathway proteins causes downstream proteins to mislocalize, revealing their order of action. Pol IVa acts first, and its localization is RNA dependent, suggesting an RNA template. We hypothesize that maintenance of the heterochromatic state involves locus-specific Pol IVa transcription followed by siRNA production and assembly of AGO4- and NRPD1b-containing silencing complexes within nucleolar processing centers

Antiviral CD8+ T cell effector activities in situ are regulated by target cell type

In the lungs of mice infected with influenza, the activity of cytotoxic T lymphocytes is modulated by the type of target cell encountered

Arousal frequency is associated with increased fatigue in obstructive sleep apnea

Fatigue is an important and often underemphasized symptom in patients with obstructive sleep apnea (OSA). Sleep fragmentation, i.e., arousals and disruptions in sleep architecture, is common in patients with OSA and may potentially contribute to their fatigue. We hypothesized that arousal frequency and changes in sleep architecture contribute to the fatigue experienced by patients with OSA. Seventy-three patients with diagnosed but untreated OSA (AHI ≥ 15) were enrolled in this study. A baseline polysomnogram was obtained, and fatigue was measured with the Multidimensional Fatigue Symptom Inventory-short form (MFSI-sf). We evaluated the association between fatigue and arousals and various polysomongraphic variables, including sleep stages and sleep efficiency. Significant correlations between MFSI-sf subscale scores and various arousal indices were noted. Emotional fatigue scores were associated with total arousal index (r = 0.416, p = .021), respiratory movement arousal index (r = 0.346, p = .025), and spontaneous movement arousal index (r = 0.378, p = .025). Physical fatigue scores were associated with total arousal index (r = 0.360, p = .033) and respiratory movement arousal index (r = 0.304, p = .040). Percent of stage 1 sleep and REM sleep were also associated with physical and emotional fatigue scores. Hierarchal linear regression analysis demonstrated that emotional fatigue scores were independently associated with spontaneous movement arousals after controlling for age, body mass index, depression, and sleep apnea severity. These findings suggest that arousals may contribute to the fatigue seen in patients with OSA

The Cell Cycle Time of CD8+ T Cells Responding In Vivo Is Controlled by the Type of Antigenic Stimulus

A hallmark of cells comprising the mammalian adaptive immune system is the requirement for these rare naïve T (and B) lymphocytes directed to a specific microorganism to undergo proliferative expansion upon first encounter with this antigen. In the case of naïve CD8+ T cells the ability of these rare quiescent lymphocytes to rapidly activate and expand into effector T cells in numbers sufficient to control viral and certain bacterial infections can be essential for survival. In this report we examined the activation, cell cycle time and initial proliferative response of naïve murine CD8+ T cells responding in vivo to Influenza and Vaccinia virus infection or vaccination with viral antigens. Remarkably, we observed that CD8+ T cells could divide and proliferate with an initial cell division time of as short as 2 hours. The initial cell cycle time of responding CD8+ T cells is not fixed but is controlled by the antigenic stimulus provided by the APC in vivo. Initial cell cycle time influences the rate of T cell expansion and the numbers of effector T cells subsequently accumulating at the site of infection. The T cell cycle time varies with duration of the G1 phase of the cell cycle. The duration of G1 is inversely correlated with the phosphorylation state of the retinoblastoma (Rb) protein in the responding T cells. The implication of these findings for the development of adaptive immune responses and the regulation of cell cycle in higher eukaryotic cells is discussed

Memory of the Vernalized State in Plants including the Model Grass Brachypodium distachyon

Plant species that have a vernalization requirement exhibit variation in the ability to remember winter—i.e., variation in the stability of the vernalized state. Studies in Arabidopsis have demonstrated that molecular memory involves changes in the chromatin state and expression of the flowering repressor FLOWERING LOCUS C, and have revealed that single-gene differences can have large effects on the stability of the vernalized state. In the perennial Arabidopsis relative Arabis alpina, the lack of memory of winter is critical for its perennial life history. Our studies of flowering behavior in the model grass Brachypodium distachyon reveal extensive variation in the vernalization requirement, and studies of a particular Brachypodium accession that has a qualitative requirement for both cold exposure and inductive daylength reveals that Brachypodium can exhibit a highly stable vernalized state

Intersection of Small RNA Pathways in Arabidopsis thaliana Sub-Nuclear Domains

In Arabidopsis thaliana, functionally diverse small RNA (smRNA) pathways bring about decreased RNA accumulation of target genes via several different mechanisms. Cytological experiments have suggested that the processing of microRNAs (miRNAs) and heterochromatic small interfering RNAs (hc-siRNAs) occurs within a specific nuclear domain that can present Cajal Body (CB) characteristics. It is unclear whether single or multiple smRNA-related domains are found within the same CB and how specialization of the smRNA pathways is determined within this specific sub-compartment. To ascertain whether nuclear smRNA centers are spatially related, we localized key proteins required for siRNA or miRNA biogenesis by immunofluorescence analysis. The intranuclear distribution of the proteins revealed that hc-siRNA, miRNA and trans-acting siRNA (ta-siRNA) pathway proteins accumulate and colocalize within a sub-nuclear structure in the nucleolar periphery. Furthermore, colocalization of miRNA- and siRNA-pathway members with CB markers, and reduced wild-type localization patterns in CB mutants indicates that proper nuclear localization of these proteins requires CB integrity. We hypothesize that these nuclear domains could be important for RNA silencing and may partially explain the functional redundancies and interactions among components of the same protein family. The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place

Nonadditive Gene Expression in Diploid and Triploid Hybrids of Maize

The molecular basis of hybrid vigor (heterosis) has remained unknown despite the importance of this phenomenon in evolution and in practical breeding programs. To formulate a molecular basis of heterosis, an understanding of gene expression in inbred and hybrid states is needed. In this study, we examined the amount of various transcripts in hybrid and inbred individuals (B73 and Mo17) to determine whether the quantities of specific messenger RNAs were additive or nonadditive in the hybrids. Further, we examined the levels of the same transcripts in hybrid triploid individuals that had received unequal genomic contributions, one haploid genome from one parent and two from the other. If allelic expression were merely the additive value in hybrids from the two parents, the midparent values would be observed. Our study revealed that a substantial number of genes do not exhibit the midparent value of expression in hybrids. Instead, transcript levels in the diploid hybrids correlate negatively with the levels in diploid inbreds. Although transcript levels were clearly nonadditive, transcript levels in triploid hybrids were affected by genomic dosage