Coupling of co-transcriptional splicing and 3' end Pol II pausing during termination in Arabidopsis.

BACKGROUND: In Arabidopsis, RNA Polymerase II (Pol II) often pauses within a few hundred base pairs downstream of the polyadenylation site, reflecting efficient transcriptional termination, but how such pausing is regulated remains largely elusive. RESULT: Here, we analyze Pol II dynamics at 3' ends by combining comprehensive experiments with mathematical modelling. We generate high-resolution serine 2 phosphorylated (Ser2P) Pol II positioning data specifically enriched at 3' ends and define a 3' end pause index (3'PI). The position but not the extent of the 3' end pause correlates with the termination window size. The 3'PI is not decreased but even mildly increased in the termination deficient mutant xrn3, indicating 3' end pause is a regulatory step early during the termination and before XRN3-mediated RNA decay that releases Pol II. Unexpectedly, 3'PI is closely associated with gene exon numbers and co-transcriptional splicing efficiency. Multiple exons genes often display stronger 3' end pauses and more efficient on-chromatin splicing than genes with fewer exons. Chemical inhibition of splicing strongly reduces the 3'PI and disrupts its correlation with exon numbers but does not globally impact 3' end readthrough levels. These results are further confirmed by fitting Pol II positioning data with a mathematical model, which enables the estimation of parameters that define Pol II dynamics. CONCLUSION: Our work highlights that the number of exons via co-transcriptional splicing is a major determinant of Pol II pausing levels at the 3' end of genes in plants.

Distinct chromatin signatures in the Arabidopsis male gametophyte.

Epigenetic reprogramming in the germline contributes to the erasure of epigenetic inheritance across generations in mammals but remains poorly characterized in plants. Here we profiled histone modifications throughout Arabidopsis male germline development. We find that the sperm cell has widespread apparent chromatin bivalency, which is established by the acquisition of H3K27me3 or H3K4me3 at pre-existing H3K4me3 or H3K27me3 regions, respectively. These bivalent domains are associated with a distinct transcriptional status. Somatic H3K27me3 is generally reduced in sperm, while dramatic loss of H3K27me3 is observed at only ~700 developmental genes. The incorporation of the histone variant H3.10 facilitates the establishment of sperm chromatin identity without a strong impact on resetting of somatic H3K27me3. Vegetative nuclei harbor thousands of specific H3K27me3 domains at repressed genes, while pollination-related genes are highly expressed and marked by gene body H3K4me3. Our work highlights putative chromatin bivalency and restricted resetting of H3K27me3 at developmental regulators as key features in plant pluripotent sperm.

ALBA proteins confer thermotolerance through stabilizing HSF messenger RNAs in cytoplasmic granules.

High temperature is one of the major environmental stresses affecting plant growth and fitness. Heat stress transcription factors (HSFs) play critical roles in regulating the expression of heat-responsive genes. However, how HSFs are regulated remains obscure. Here, we show that ALBA4, ALBA5 and ALBA6, which phase separate into stress granules (SGs) and processing bodies (PBs) under heat stress, directly bind selected messenger RNAs, including HSF mRNAs, and recruit them into SGs and PBs to protect them from degradation under heat stress in Arabidopsis. The alba456 triple mutants, but not single and double mutants, display pleiotropic developmental defects and hypersensitivity to heat stress. Mutations in XRN4, a cytoplasmic 5' to 3' exoribonuclease, can rescue the observed developmental and heat-sensitive phenotypes of alba456 seedlings. Our study reveals a new layer of regulation for HSFs whereby HSF mRNAs are stabilized by redundant action of ALBA proteins in SGs and PBs for plant thermotolerance.

Recombinase-mediated gene stacking in cotton.

Site-specific gene stacking could reduce the number of segregating loci and expedite the introgression of transgenes from experimental lines to field lines. Recombinase-mediated site-specific gene stacking provides a flexible and efficient solution, but this approach requires a recombinase recognition site in the genome. Here, we describe several cotton (Gossypium hirsutum cv. Coker 312) target lines suitable for Mycobacteriophage Bxb1 recombinase-mediated gene stacking. Obtained through the empirical screening of random insertion events, each of these target lines contains a single intact copy of the target construct with precise sequences of RS2, lox, and attP sites that is not inserted within or close to a known gene or near a centromere and shows good expression of the reporter gene gfp. Gene stacking was tested with insertion of different combinations of three candidate genes for resistance to verticillium wilt into three cotton target lines: CTS1, CTS3, and CTS4. Nine site-specific integration events were recovered from 95 independently transformed embryogenic calluses. Southern and DNA sequence analyses of regenerated plants confirmed precise site-specific integration, and resistance to verticillium wilt was observed for plant CTS1i3, which has a single precise copy of site-specifically integrated DNA. These cotton target lines can serve as foundation lines for recombinase-mediated gene stacking to facilitate precise DNA integration and introgression to field cultivars.

Mechanistic Study of Copper-Catalyzed C-H Hydroxylation/C-S Coupling by ESI-HR MS and DFT Calculations.

The reaction mechanism of Cu-catalyzed C-H hydroxylation/C-S coupling was studied using electrospray ionization high resolution mass spectrometry (ESI-HR MS) and density functional theory calculations (DFT). Notably, a series of CuI and CuIII complexes were observed as key intermediates and identified using ESI-HR MS. Furthermore, a catalyst cycle involving proton abstraction/oxidative addition/reductive elimination was proposed. This study is important and valuable with respect to C-H functionalization.