The RALF signaling pathway regulates cell wall integrity during pollen tube growth in maize.

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis (Arabidopsis thaliana). To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize (Zea mays). We show that Clade IB ZmRALF2/3 mutations, but not Clade III ZmRALF1/5 mutations, cause cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located in the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution patterns leading to altered cell wall organization and thickness culminating in pollen tube burst. Clade IB, but not Clade III ZmRALFs, strongly interact as ligands with the pollen-specific Catharanthus roseus RLK1-like (CrRLK1L) receptor kinases Z. mays FERONIA-like (ZmFERL) 4/7/9, LORELEI-like glycosylphosphatidylinositol-anchor (LLG) proteins Z. mays LLG 1 and 2 (ZmLLG1/2), and Z. mays pollen extension-like (PEX) cell wall proteins ZmPEX2/4. Notably, ZmFERL4 outcompetes ZmLLG2 and ZmPEX2 outcompetes ZmFERL4 for ZmRALF2 binding. Based on these data, we suggest that Clade IB RALFs act in a dual role as cell wall components and extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in maize and probably other plants.

Nucleosomes Stabilize ssRNA-dsDNA Triple Helices in Human Cells.

Chromatin-associated non-coding RNAs modulate the epigenetic landscape and its associated gene expression program. The formation of triple helices is one mechanism of sequence-specific targeting of RNA to chromatin. With this study, we show an important role of the nucleosome and its relative positioning to the triplex targeting site (TTS) in stabilizing RNA-DNA triplexes in vitro and in vivo. Triplex stabilization depends on the histone H3 tail and the location of the TTS close to the nucleosomal DNA entry-exit site. Genome-wide analysis of TTS-nucleosome arrangements revealed a defined chromatin organization with an enrichment of arrangements that allow triplex formation at active regulatory sites and accessible chromatin. We further developed a method to monitor nucleosome-RNA triplexes in vivo (TRIP-seq), revealing RNA binding to TTS sites adjacent to nucleosomes. Our data strongly support an activating role for RNA triplex-nucleosome complexes, pinpointing triplex-mediated epigenetic regulation in vivo.

Characterization of the nuclear import of the human CHD4-NuRD complex.

Chromatin remodeling enzymes form large multiprotein complexes that play central roles in regulating access to the genome. Here, we characterize the nuclear import of the human CHD4 protein. We show that CHD4 enters the nucleus by means of several importin-α proteins (1, 5, 6 and 7), but independently of importin ß1. Importin α1 directly interacts with a monopartite 'KRKR'-motif in the N-terminus of CHD4 (amino acids 304-307). However, alanine mutagenesis of this motif only leads to an ∼50% reduction in nuclear localization of CHD4, implying that there are additional import mechanisms. Interestingly, we could show that CHD4 was already associated with the nucleosome remodeling deacetylase (NuRD) core subunits, such as MTA2, HDAC1 and RbAp46 (also known as RBBP7), in the cytoplasm, suggesting an assembly of the NuRD core complex before nuclear import. We propose that, in addition to the importin-α-dependent nuclear localization signal, CHD4 is dragged into the nucleus by a 'piggyback' mechanism using the import signals of the associated NuRD subunits.

Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Nrf2 pre-recruitment at Enhancer 2 is a hallmark of H2 O2 -induced epigenetic transcriptional memory in the HMOX1 gene in human umbilical artery endothelial cells.

Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity.

Arabidopsis mRNA export factor MOS11: molecular interactions and role in abiotic stress responses.

Transcription and export (TREX) is a multi-subunit complex that links synthesis, processing and export of mRNAs. It interacts with the RNA helicase UAP56 and export factors such as MOS11 and ALYs to facilitate nucleocytosolic transport of mRNAs. Plant MOS11 is a conserved, but sparsely researched RNA-binding export factor, related to yeast Tho1 and mammalian CIP29/SARNP. Using biochemical approaches, the domains of Arabidopsis thaliana MOS11 required for interaction with UAP56 and RNA-binding were identified. Further analyses revealed marked genetic interactions between MOS11 and ALY genes. Cell fractionation in combination with transcript profiling demonstrated that MOS11 is required for export of a subset of mRNAs that are shorter and more GC-rich than MOS11-independent transcripts. The central α-helical domain of MOS11 proved essential for physical interaction with UAP56 and for RNA-binding. MOS11 is involved in the nucleocytosolic transport of mRNAs that are upregulated under stress conditions and accordingly mos11 mutant plants turned out to be sensitive to elevated NaCl concentrations and heat stress. Collectively, our analyses identify functional interaction domains of MOS11. In addition, the results establish that mRNA export is critically involved in the plant response to stress conditions and that MOS11 plays a prominent role at this.

Phosphorylation of the FACT histone chaperone subunit SPT16 affects chromatin at RNA polymerase II transcriptional start sites in Arabidopsis.

The heterodimeric histone chaperone FACT, consisting of SSRP1 and SPT16, contributes to dynamic nucleosome rearrangements during various DNA-dependent processes including transcription. In search of post-translational modifications that may regulate the activity of FACT, SSRP1 and SPT16 were isolated from Arabidopsis cells and analysed by mass spectrometry. Four acetylated lysine residues could be mapped within the basic C-terminal region of SSRP1, while three phosphorylated serine/threonine residues were identified in the acidic C-terminal region of SPT16. Mutational analysis of the SSRP1 acetylation sites revealed only mild effects. However, phosphorylation of SPT16 that is catalysed by protein kinase CK2, modulates histone interactions. A non-phosphorylatable version of SPT16 displayed reduced histone binding and proved inactive in complementing the growth and developmental phenotypes of spt16 mutant plants. In plants expressing the non-phosphorylatable SPT16 version we detected at a subset of genes enrichment of histone H3 directly upstream of RNA polymerase II transcriptional start sites (TSSs) in a region that usually is nucleosome-depleted. This suggests that some genes require phosphorylation of the SPT16 acidic region for establishing the correct nucleosome occupancy at the TSS of active genes.

The Long Non-Coding RNA MALAT1 Modulates NR4A1 Expression through a Downstream Regulatory Element in Specific Cancer Cell Types.

Long non-coding RNAs (lncRNAs) have been shown to modulate gene expression and are involved in the initiation and progression of various cancer types. Despite the wealth of studies describing transcriptome changes upon lncRNA knockdown, there is limited information describing lncRNA-mediated effects on regulatory elements (REs) modulating gene expression. In this study, we investigated how the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA regulates primary target genes using time-resolved MALAT1 knockdown followed by parallel RNA-seq and ATAC-seq assays. The results revealed that MALAT1 primarily regulates specific protein-coding genes and a substantial decrease in the accessibility downstream of the NR4A1 gene that was associated with a decreased NR4A1 expression. Moreover, the presence of an NR4A1-downstream RE was demonstrated by CRISPR-i assays to define a functional MALAT1/NR4A1 axis. By analyzing TCGA data, we identified a positive correlation between NR4A1 expression and NR4A1-downstream RE accessibility in breast cancer but not in pancreatic cancer. Accordingly, this regulatory mechanism was experimentally validated in breast cancer cells (MCF7) but not in pancreatic duct epithelial carcinoma (PANC1) cells. Therefore, our results demonstrated that MALAT1 is involved in a molecular mechanism that fine-tunes NR4A1 expression by modulating the accessibility of a downstream RE in a cell type-specific manner.

The chromatin - triple helix connection.

Mammalian genomes are extensively transcribed, producing a large number of coding and non-coding transcripts. A large fraction of the nuclear RNAs is physically associated with chromatin, functioning in gene activation and silencing, shaping higher-order genome organisation, such as involvement in long-range enhancer-promoter interactions, transcription hubs, heterochromatin, nuclear bodies and phase transitions. Different mechanisms allow the tethering of these chromatin-associated RNAs (caRNA) to chromosomes, including RNA binding proteins, the RNA polymerases and R-loops. In this review, we focus on the sequence-specific targeting of RNA to DNA by forming triple helical structures and describe its interplay with chromatin. It turns out that nucleosome positioning at triple helix target sites and the nucleosome itself are essential factors in determining the formation and stability of triple helices. The histone H3-tail plays a critical role in triple helix stabilisation, and the role of its epigenetic modifications in this process is discussed.

Elongation factor 1 is a component of the Arabidopsis RNA polymerase II elongation complex and associates with a subset of transcribed genes.

Elongation factors modulate the efficiency of mRNA synthesis by RNA polymerase II (RNAPII) in the context of chromatin, thus contributing to implement proper gene expression programmes. The zinc-finger protein elongation factor 1 (ELF1) is a conserved transcript elongation factor (TEF), whose molecular function so far has not been studied in plants. Using biochemical approaches, we examined the interaction of Arabidopsis ELF1 with DNA and histones in vitro and with the RNAPII elongation complex in vivo. In addition, cytological assays demonstrated the nuclear localisation of the protein, and by means of double-mutant analyses, interplay with genes encoding other elongation factors was explored. The genome-wide distribution of ELF1 was addressed by chromatin immunoprecipitation. ELF1 isolated from Arabidopsis cells robustly copurified with RNAPII and various other elongation factors including SPT4-SPT5, SPT6, IWS1, FACT and PAF1C. Analysis of a CRISPR-Cas9-mediated gene editing mutant of ELF1 revealed distinct genetic interactions with mutants deficient in other elongation factors. Moreover, ELF1 associated with genomic regions actively transcribed by RNAPII. However, ELF1 occupied only c. 33% of the RNAPII transcribed loci with preference for inducible rather than constitutively expressed genes. Collectively, these results establish that Arabidopsis ELF1 shares several characteristic attributes with RNAPII TEFs.

Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators.

HISTONE MONOUBIQUITINATION1 (HUB1) and its paralog HUB2 act in a conserved heterotetrameric complex in the chromatin-mediated transcriptional modulation of developmental programs, such as flowering time, dormancy, and the circadian clock. The KHD1 and SPEN3 proteins were identified as interactors of the HUB1 and HUB2 proteins with in vitro RNA-binding activity. Mutants in SPEN3 and KHD1 had reduced rosette and leaf areas. Strikingly, in spen3 mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the hub1-4 mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and FLOWERING LOCUS C (FLC) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the spen3-1 and hub1-4 mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the CCA1α and CCA1ß splice forms were altered, and the CCA1 expression and H2Bub levels were reduced. In the spen3-1 mutant, the delay in flowering time was correlated with an enhanced FLC expression, possibly due to an increased distal versus proximal ratio of its antisense COOLAIR transcript. Together with transcriptomic and double-mutant analyses, our data revealed that the HUB1 interaction with SPEN3 links H2Bub during transcript elongation with pre-mRNA processing at CCA1 Furthermore, the presence of an intact HUB1 at the FLC is required for SPEN3 function in the formation of the FLC-derived antisense COOLAIR transcripts.

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation.

The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein-protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed ß propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.

RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure.

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I-associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I-specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

Antibody-targeted chromatin enables effective intracellular delivery and functionality of CRISPR/Cas9 expression plasmids.

We report a novel system for efficient and specific targeted delivery of large nucleic acids to and into cells. Plasmid DNA and core histones were assembled to chromatin by salt gradient dialysis and subsequently connected to bispecific antibody derivatives (bsAbs) via a nucleic acid binding peptide bridge. The resulting reconstituted vehicles termed 'plasmid-chromatin' deliver packaged nucleic acids to and into cells expressing antigens that are recognized by the bsAb, enabling intracellular functionality without detectable cytotoxicity. High efficiency of intracellular nucleic acid delivery is revealed by intracellular expression of plasmid encoded genes in most (∼90%) target cells to which the vehicles were applied under normal growth/medium conditions in nanomolar concentrations. Specific targeting, uptake and transgene expression depends on antibody-mediated cell surface binding: plasmid chromatin of identical composition but with non-targeting bsAbs or without bsAbs is ineffective. Examples that demonstrate applicability, specificity and efficacy of antibody-targeted plasmid chromatin include reporter gene constructs as well as plasmids that enable CRISPR/Cas9 mediated genome editing of target cells.

Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin.

Packaging of DNA into chromatin regulates DNA accessibility and consequently all DNA-dependent processes. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This organization has been recently questioned using microscopy techniques, proposing an irregular structure. To address the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. We assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions, performed statistical analyses and computer modelling. We found no difference in MNase accessibility, suggesting no difference in fiber folding between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially organized domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

Peculiarities of Plasmodium falciparum Gene Regulation and Chromatin Structure.

The highly complex life cycle of the human malaria parasite, Plasmodium falciparum, is based on an orchestrated and tightly regulated gene expression program. In general, eukaryotic transcription regulation is determined by a combination of sequence-specific transcription factors binding to regulatory DNA elements and the packaging of DNA into chromatin as an additional layer. The accessibility of regulatory DNA elements is controlled by the nucleosome occupancy and changes of their positions by an active process called nucleosome remodeling. These epigenetic mechanisms are poorly explored in P. falciparum. The parasite genome is characterized by an extraordinarily high AT-content and the distinct architecture of functional elements, and chromatin-related proteins also exhibit high sequence divergence compared to other eukaryotes. Together with the distinct biochemical properties of nucleosomes, these features suggest substantial differences in chromatin-dependent regulation. Here, we highlight the peculiarities of epigenetic mechanisms in P. falciparum, addressing chromatin structure and dynamics with respect to their impact on transcriptional control. We focus on the specialized chromatin remodeling enzymes and discuss their essential function in P. falciparum gene regulation.

Elucidation of the functional roles of the Q and I motifs in the human chromatin-remodeling enzyme BRG1.

The Snf2 proteins, comprising 53 different enzymes in humans, belong to the SF2 family. Many Snf2 enzymes possess chromatin-remodeling activity, requiring a functional ATPase domain consisting of conserved motifs named Q and I-VII. These motifs form two recA-like domains, creating an ATP-binding pocket. Little is known about the function of the conserved motifs in chromatin-remodeling enzymes. Here, we characterized the function of the Q and I (Walker I) motifs in hBRG1 (SMARCA4). The motifs are in close proximity to the bound ATP, suggesting a role in nucleotide binding and/or hydrolysis. Unexpectedly, when substituting the conserved residues Gln758 (Q motif) or Lys785 (I motif) of both motifs, all variants still bound ATP and exhibited basal ATPase activity similar to that of wildtype BRG1 (wtBRG1). However, all mutants lost the nucleosome-dependent stimulation of the ATPase domain. Their chromatin-remodeling rates were impaired accordingly, but nucleosome binding was retained and still comparable with that of wtBRG1. Interestingly, a cancer-relevant substitution, L754F (Q motif), displayed defects similar to the Gln758 variant(s), arguing for a comparable loss of function. Because we excluded a mutual interference of ATP and nucleosome binding, we postulate that both motifs stimulate the ATPase and chromatin-remodeling activities upon binding of BRG1 to nucleosomes, probably via allosteric mechanisms. Furthermore, mutations of both motifs similarly affect the enzymatic functionality of BRG1 in vitro and in living cells. Of note, in BRG1-deficient H1299 cells, exogenously expressed wtBRG1, but not BRG1 Q758A and BRG1 K785R, exhibited a tumor suppressor-like function.

Purine- and pyrimidine-triple-helix-forming oligonucleotides recognize qualitatively different target sites at the ribosomal DNA locus.

Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNA targeting to chromatin. Based on the rules of Hoogsteen base-pairing, polypurine sequences of a duplex can potentially form triplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyses have revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers, suggesting a potential function of RNA-DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated the potential of different sequences of human and mouse ribosomal RNA genes (rDNA) to form triplexes at different salt and pH conditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes with high affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) are more stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differential sequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G-C content of the TTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, shows that in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predict genomic TTS.

The UAP56-Interacting Export Factors UIEF1 and UIEF2 Function in mRNA Export.

In eukaryotes, the regulated transport of mRNAs from the nucleus to the cytosol through nuclear pore complexes represents an important step in the expression of protein-coding genes. In plants, the mechanism of nucleocytosolic mRNA transport and the factors involved are poorly understood. The Arabidopsis (Arabidopsis thaliana) genome encodes two likely orthologs of UAP56-interacting factor, which acts as mRNA export factor in mammalian cells. In yeast and plant cells, both proteins interact directly with the mRNA export-related RNA helicase UAP56 and the interaction was mediated by an N-terminal UAP56-binding motif. Accordingly, the two proteins were termed UAP56-INTERACTING EXPORT FACTOR1 and 2 (UIEF1/2). Despite lacking a known RNA-binding motif, recombinant UIEF1 interacted with RNA, and the C-terminal part of UIEF1 mainly contributed to the RNA interaction. Mutation of UIEF1, UIEF2, or both in the double-mutant 2xuief caused modest growth defects. A cross between the 2xuief and 4xaly (defective in the four ALY1-4 mRNA export factors) mutants produced the sextuple mutant 4xaly 2xuief, which displayed more severe growth impairment than the 4xaly plants. Developmental defects including delayed bolting and reduced seed set were observed in the 4xaly but not the 2xuief plants. Analysis of the cellular distribution of polyadenylated mRNAs revealed more pronounced nuclear mRNA accumulation in 4xaly 2xuief than in 2xuief and 4xaly cells. In conclusion, the results indicate that UIEF1 and UIEF2 act as mRNA export factors in plants and that they cooperate with ALY1-ALY4 to mediate efficient nucleocytosolic mRNA transport.

The Composition of the Arabidopsis RNA Polymerase II Transcript Elongation Complex Reveals the Interplay between Elongation and mRNA Processing Factors.

Transcript elongation factors (TEFs) are a heterogeneous group of proteins that control the efficiency of transcript elongation of subsets of genes by RNA polymerase II (RNAPII) in the chromatin context. Using reciprocal tagging in combination with affinity purification and mass spectrometry, we demonstrate that in Arabidopsis thaliana, the TEFs SPT4/SPT5, SPT6, FACT, PAF1-C, and TFIIS copurified with each other and with elongating RNAPII, while P-TEFb was not among the interactors. Additionally, NAP1 histone chaperones, ATP-dependent chromatin remodeling factors, and some histone-modifying enzymes including Elongator were repeatedly found associated with TEFs. Analysis of double mutant plants defective in different combinations of TEFs revealed genetic interactions between genes encoding subunits of PAF1-C, FACT, and TFIIS, resulting in synergistic/epistatic effects on plant growth/development. Analysis of subnuclear localization, gene expression, and chromatin association did not provide evidence for an involvement of the TEFs in transcription by RNAPI (or RNAPIII). Proteomics analyses also revealed multiple interactions between the transcript elongation complex and factors involved in mRNA splicing and polyadenylation, including an association of PAF1-C with the polyadenylation factor CstF. Therefore, the RNAPII transcript elongation complex represents a platform for interactions among different TEFs, as well as for coordinating ongoing transcription with mRNA processing.