Different elongation factors distinctly modulate RNA polymerase II transcription in Arabidopsis.

Various transcript elongation factors (TEFs) including modulators of RNA polymerase II (RNAPII) activity and histone chaperones tune the efficiency of transcription in the chromatin context. TEFs are involved in establishing gene expression patterns during growth and development in Arabidopsis, while little is known about the genomic distribution of the TEFs and the way they facilitate transcription. We have mapped the genome-wide occupancy of the elongation factors SPT4-SPT5, PAF1C and FACT, relative to that of elongating RNAPII phosphorylated at residues S2/S5 within the carboxyterminal domain. The distribution of SPT4-SPT5 along transcribed regions closely resembles that of RNAPII-S2P, while the occupancy of FACT and PAF1C is rather related to that of RNAPII-S5P. Under transcriptionally challenging heat stress conditions, mutant plants lacking the corresponding TEFs are differentially impaired in transcript synthesis. Strikingly, in plants deficient in PAF1C, defects in transcription across intron/exon borders are observed that are cumulative along transcribed regions. Upstream of transcriptional start sites, the presence of FACT correlates with nucleosomal occupancy. Under stress conditions FACT is particularly required for transcriptional upregulation and to promote RNAPII transcription through +1 nucleosomes. Thus, Arabidopsis TEFs are differently distributed along transcribed regions, and are distinctly required during transcript elongation especially upon transcriptional reprogramming.

Transcript elongation by RNA polymerase II in plants: factors, regulation and impact on gene expression.

Transcriptional elongation by RNA polymerase II (RNAPII) through chromatin is a dynamic and highly regulated step of eukaryotic gene expression. A combination of transcript elongation factors (TEFs) including modulators of RNAPII activity and histone chaperones facilitate efficient transcription on nucleosomal templates. Biochemical and genetic analyses, primarily performed in Arabidopsis, provided insight into the contribution of TEFs to establish gene expression patterns during plant growth and development. In addition to summarising the role of TEFs in plant gene expression, we emphasise in our review recent advances in the field. Thus, mechanisms are presented how aberrant intragenic transcript initiation is suppressed by repressing transcriptional start sites within coding sequences. We also discuss how transcriptional interference of ongoing transcription with neighbouring genes is prevented. Moreover, it appears that plants make no use of promoter-proximal RNAPII pausing in the way mammals do, but there are nucleosome-defined mechanism(s) that determine the efficiency of mRNA synthesis by RNAPII. Accordingly, a still growing number of processes related to plant growth, development and responses to changing environmental conditions prove to be regulated at the level of transcriptional elongation.

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.

Cytosolic RGG RNA-binding proteins are temperature sensitive flowering time regulators in Arabidopsis.

mRNA translation is tightly regulated by various classes of RNA-binding proteins (RBPs) during development and in response to changing environmental conditions. In this study, we characterize the arginine-glycine-glycine (RGG) motif containing RBP family of Arabidopsis thaliana representing homologues of the multifunctional translation regulators and ribosomal preservation factors Stm1 from yeast (ScStm1) and human SERBP1 (HsSERBP1). The Arabidopsis genome encodes three RGG proteins named AtRGGA, AtRGGB and AtRGGC. While AtRGGA is ubiquitously expressed, AtRGGB and AtRGGC are enriched in dividing cells. All AtRGGs localize almost exclusively to the cytoplasm and bind with high affinity to ssRNA, while being capable to interact with most nucleic acids, except dsRNA. A protein-interactome study shows that AtRGGs interact with ribosomal proteins and proteins involved in RNA processing and transport. In contrast to ScStm1, AtRGGs are enriched in ribosome-free fractions in polysome profiles, suggesting additional plant-specific functions. Mutant studies show that AtRGG proteins differentially regulate flowering time, with a distinct and complex temperature dependency for each AtRGG protein. In conclusion, we suggest that AtRGGs function in fine-tuning translation efficiency to control flowering time and potentially other developmental processes in response to environmental changes.

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.

Critical Role of Transcript Cleavage in Arabidopsis RNA Polymerase II Transcriptional Elongation.

Transcript elongation factors associate with elongating RNA polymerase II (RNAPII) to control the efficiency of mRNA synthesis and consequently modulate plant growth and development. Encountering obstacles during transcription such as nucleosomes or particular DNA sequences may cause backtracking and transcriptional arrest of RNAPII. The elongation factor TFIIS stimulates the intrinsic transcript cleavage activity of the polymerase, which is required for efficient rescue of backtracked/arrested RNAPII. A TFIIS mutant variant (TFIISmut) lacks the stimulatory activity to promote RNA cleavage, but instead efficiently inhibits unstimulated transcript cleavage by RNAPII. We could not recover viable Arabidopsis (Arabidopsis thaliana) tfIIs plants constitutively expressing TFIISmut. Induced, transient expression of TFIISmut in tfIIs plants provoked severe growth defects, transcriptomic changes and massive, transcription-related redistribution of elongating RNAPII within transcribed regions toward the transcriptional start site. The predominant site of RNAPII accumulation overlapped with the +1 nucleosome, suggesting that upon inhibition of RNA cleavage activity, RNAPII arrest prevalently occurs at this position. In the presence of TFIISmut, the amount of RNAPII was reduced, which could be reverted by inhibiting the proteasome, indicating proteasomal degradation of arrested RNAPII. Our findings suggest that polymerase backtracking/arrest frequently occurs in plant cells, and RNAPII-reactivation is essential for correct transcriptional output and proper growth/development.

Nuclear export of plant pararetrovirus mRNAs involves the TREX complex, two viral proteins and the highly structured 5' leader region.

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.

The transcription and export complex THO/TREX contributes to transcription termination in plants.

Transcription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3'UTR extensions in several endogenous genes. These results demonstrate that THO/TREX complex contributes to the regulation of transcription termination.

H3K9 demethylases IBM1 and JMJ27 are required for male meiosis in Arabidopsis thaliana.

Dimethylation of histone H3 lysine 9 (H3K9me2), a crucial modification for heterochromatin formation and transcriptional silencing, is essential for proper meiotic prophase progression in mammals. We analyzed meiotic defects and generated genome-wide profiles of H3K9me2 and transcriptomes for the mutants of H3K9 demethylases. Moreover, we also identified proteins interacting with H3K9 demethylases. H3K9me2 is usually found at transposable elements and repetitive sequences but is absent from the bodies of protein-coding genes. In this study, we show that the Arabidopsis thaliana H3K9 demethylases IBM1 and JMJ27 cooperatively regulate crossover formation and chromosome segregation. They protect thousands of protein-coding genes from ectopic H3K9me2, including genes essential for meiotic prophase progression. In addition to removing H3K9me2, IBM1 and JMJ27 interact with the Precocious Dissociation of Sisters 5 (PDS5) cohesin complex cofactors. The pds5 mutant shared similar transcriptional alterations with ibm1 jmj27, including meiosis-essential genes, yet without affecting H3K9me2 levels. Hence, PDS5s, together with IBM1 and JMJ27, regulate male meiosis and gene expression independently of H3K9 demethylation. These findings uncover a novel role of H3K9me2 removal in meiosis and a new function of H3K9 demethylases and cohesin cofactors in meiotic transcriptional regulation.

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 Arabidopsis condensin CAP-D subunits arrange interphase chromatin.

Condensins are best known for their role in shaping chromosomes. Other functions such as organizing interphase chromatin and transcriptional control have been reported in yeasts and animals, but little is known about their function in plants. To elucidate the specific composition of condensin complexes and the expression of CAP-D2 (condensin I) and CAP-D3 (condensin II), we performed biochemical analyses in Arabidopsis. The role of CAP-D3 in interphase chromatin organization and function was evaluated using cytogenetic and transcriptome analysis in cap-d3 T-DNA insertion mutants. CAP-D2 and CAP-D3 are highly expressed in mitotically active tissues. In silico and pull-down experiments indicate that both CAP-D proteins interact with the other condensin I and II subunits. In cap-d3 mutants, an association of heterochromatic sequences occurs, but the nuclear size and the general histone and DNA methylation patterns remain unchanged. Also, CAP-D3 influences the expression of genes affecting the response to water, chemicals, and stress. The expression and composition of the condensin complexes in Arabidopsis are similar to those in other higher eukaryotes. We propose a model for the CAP-D3 function during interphase in which CAP-D3 localizes in euchromatin loops to stiffen them and consequently separates centromeric regions and 45S rDNA repeats.

Targeted Recruitment of the Basal Transcriptional Machinery by LNK Clock Components Controls the Circadian Rhythms of Nascent RNAs in Arabidopsis.

The rhythms of steady-state mRNA expression pervade nearly all circadian systems. However, the mechanisms behind the rhythmic transcriptional synthesis and its correlation with circadian expression remain fully unexplored, particularly in plants. Here, we discovered a multifunctional protein complex that orchestrates the rhythms of transcriptional activity in Arabidopsis thaliana The expression of the circadian oscillator genes TIMING OF CAB EXPRESSION1/PSEUDO-RESPONSE REGULATOR1 and PSEUDO-RESPONSE REGULATOR5 initially relies on the modular function of the clock-related factor REVEILLE8: its MYB domain provides the DNA binding specificity, while its LCL domain recruits the clock components, NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED proteins (LNKs), to target promoters. LNKs, in turn, specifically interact with RNA Polymerase II and the transcript elongation FACT complex to rhythmically co-occupy the target loci. The functional interaction of these components is central for chromatin status, transcript initiation, and elongation as well as for proper rhythms in nascent RNAs. Thus, our findings explain how genome readout of environmental information ultimately results in rhythmic changes of gene expression.

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.

ALY RNA-Binding Proteins Are Required for Nucleocytosolic mRNA Transport and Modulate Plant Growth and Development.

The regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step linking transcript synthesis and processing with translation. However, in plants, only a few of the factors that act in the mRNA export pathway have been functionally characterized. Flowering plant genomes encode several members of the ALY protein family, which function as mRNA export factors in other organisms. Arabidopsis (Arabidopsis thaliana) ALY1 to ALY4 are commonly detected in root and leaf cells, but they are differentially expressed in reproductive tissue. Moreover, the subnuclear distribution of ALY1/2 differs from that of ALY3/4. ALY1 binds with higher affinity to single-stranded RNA than double-stranded RNA and single-stranded DNA and interacts preferentially with 5-methylcytosine-modified single-stranded RNA. Compared with the full-length protein, the individual RNA recognition motif of ALY1 binds RNA only weakly. ALY proteins interact with the RNA helicase UAP56, indicating a link to the mRNA export machinery. Consistently, ALY1 complements the lethal phenotype of yeast cells lacking the ALY1 ortholog Yra1. Whereas individual aly mutants have a wild-type appearance, disruption of ALY1 to ALY4 in 4xaly plants causes vegetative and reproductive defects, including strongly reduced growth, altered flower morphology, as well as abnormal ovules and female gametophytes, causing reduced seed production. Moreover, polyadenylated mRNAs accumulate in the nuclei of 4xaly cells. Our results highlight the requirement of efficient mRNA nucleocytosolic transport for proper plant growth and development and indicate that ALY1 to ALY4 act partly redundantly in this process; however, differences in expression and subnuclear localization suggest distinct functions.

Nucleocytosolic mRNA transport in plants: export factors and their influence on growth and development.

In eukaryotes, the regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step in the expression of protein-coding genes, as it links nuclear mRNA synthesis with cytosolic translation. The pre-mRNAs that are synthesised by RNA polymerase II are processed by 5´-capping, splicing, and 3´-polyadenylation. The multi-subunit THO/TREX complex integrates mRNA biogenesis with their nucleocytosolic transport. Various export factors are recruited to the mRNAs during their maturation, which occurs essentially co-transcriptionally. These RNA-bound export factors ensure efficient transport of the export-competent mRNAs through nuclear pore complexes. In recent years, several factors involved in plant mRNA export have been functionally characterised. Analysis of mutant plants has demonstrated that impaired mRNA export causes defects in growth and development. Moreover, there is accumulating evidence that mRNA export can influence processes such as plant immunity, circadian regulation, and stress responses. Therefore, it is important to learn more details about the mechanism of nucleocytosolic mRNA transport in plants and its physiological significance.

The Arabidopsis histone chaperone FACT is required for stress-induced expression of anthocyanin biosynthetic genes.

KEY MESSAGE: The histone chaperone FACT is involved in the expression of genes encoding anthocyanin biosynthetic enzymes also upon induction by moderate high-light and therefore contributes to the stress-induced plant pigmentation. The histone chaperone FACT consists of the SSRP1 and SPT16 proteins and associates with transcribing RNAPII (RNAPII) along the transcribed region of genes. FACT can promote transcriptional elongation by destabilising nucleosomes in the path of RNA polymerase II, thereby facilitating efficient transcription of chromatin templates. Transcript profiling of Arabidopsis plants depleted in SSRP1 or SPT16 demonstrates that only a small subset of genes is differentially expressed relative to wild type. The majority of these genes is either up- or down-regulated in both the ssrp1 and spt16 plants. Among the down-regulated genes, those encoding enzymes of the biosynthetic pathway of the plant secondary metabolites termed anthocyanins (but not regulators of the pathway) are overrepresented. Upon exposure to moderate high-light stress several of these genes are up-regulated to a lesser extent in ssrp1/spt16 compared to wild type plants, and accordingly the mutant plants accumulate lower amounts of anthocyanin pigments. Moreover, the expression of SSRP1 and SPT16 is induced under these conditions. Therefore, our findings indicate that FACT is a novel factor required for the accumulation of anthocyanins in response to light-induction.

The Arabidopsis THO/TREX component TEX1 functionally interacts with MOS11 and modulates mRNA export and alternative splicing events.

KEY MESSAGE: We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development. TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.

The transcript elongation factor SPT4/SPT5 is involved in auxin-related gene expression in Arabidopsis.

The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4-RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes. In SPT4-RNAi plants, elevated levels of RNAPII and SPT5 are detected within transcribed regions (including those of downregulated genes), indicating transcript elongation defects in these plants. Therefore, SPT4/SPT5 acts as a TEF in Arabidopsis, regulating transcription during the elongation stage with particular impact on the expression of certain auxin-related genes.