Targeting Spt5-Pol II by Small-Molecule Inhibitors Uncouples Distinct Activities and Reveals Additional Regulatory Roles.

Spt5 is a conserved and essential transcription elongation factor that promotes promoter-proximal pausing, promoter escape, elongation, and mRNA processing. Spt5 plays specific roles in the transcription of inflammation and stress-induced genes and tri-nucleotide expanded-repeat genes involved in inherited neurological pathologies. Here, we report the identification of Spt5-Pol II small-molecule inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on promoter-proximal pausing, NF-κB activation, and expanded-repeat huntingtin gene transcription. Using SPIs, we identified Spt5 target genes that responded with profoundly diverse kinetics. SPIs uncovered the regulatory role of Spt5 in metabolism via GDF15, a food intake- and body weight-inhibitory hormone. SPIs further unveiled a role for Spt5 in promoting the 3' end processing of histone genes. While several SPIs affect all Spt5 functions, a few inhibit a single one, implying uncoupling and selective targeting of Spt5 activities. SPIs expand the understanding of Spt5-Pol II functions and are potential drugs against metabolic and neurodegenerative diseases.

Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes.

Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (HTT), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). Here we report that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.

The Paf1 complex is required for RNA polymerase II removal in response to DNA damage.

Rpb1, the largest subunit of RNA polymerase II (RNAPII), is rapidly polyubiquitinated and degraded in response to DNA damage; this process is considered to be a "mechanism of last resort'' employed by cells. The underlying mechanism of this process remains elusive. Here, we uncovered a previously uncharacterized multistep pathway in which the polymerase-associated factor 1 (Paf1) complex (PAF1C, composed of the subunits Ctr9, Paf1, Leo1, Cdc73, and Rtf1) is involved in regulating the RNAPII pool by stimulating Elongin-Cullin E3 ligase complex-mediated Rpb1 polyubiquitination and subsequent degradation by the proteasome following DNA damage. Mechanistically, Spt5 is dephosphorylated following DNA damage, thereby weakening the interaction between the Rtf1 subunit and Spt5, which might be a key step in initiating Rpb1 degradation. Next, Rad26 is loaded onto stalled RNAPII to replace the Spt4/Spt5 complex in an RNAPII-dependent manner and, in turn, recruits more PAF1C to DNA lesions via the binding of Rad26 to the Leo1 subunit. Importantly, the PAF1C, assembled in a Ctr9-mediated manner, coordinates with Rad26 to localize the Elongin-Cullin complex on stalled RNAPII, thereby inducing RNAPII removal, in which the heterodimer Paf1/Leo1 and the subunit Cdc73 play important roles. Together, our results clearly revealed a new role of the intact PAF1C in regulating the RNAPII pool in response to DNA damage.

Rpb7 represses transcription-coupled nucleotide excision repair.

Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) that is regulated by multiple facilitators, such as Rad26, and repressors, such as Rpb4 and Spt4/Spt5. How these factors interplay with each other and with core RNA polymerase II (RNAPII) remains largely unknown. In this study, we identified Rpb7, an essential RNAPII subunit, as another TCR repressor and characterized its repression of TCR in the AGP2, RPB2, and YEF3 genes, which are transcribed at low, moderate, and high rates, respectively. The Rpb7 region that interacts with the KOW3 domain of Spt5 represses TCR largely through the same common mechanism as Spt4/Spt5, as mutations in this region mildly enhance the derepression of TCR by spt4Δ only in the YEF3 gene but not in the AGP2 or RPB2 gene. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII repress TCR largely independently of Spt4/Spt5, as mutations in these regions synergistically enhance the derepression of TCR by spt4Δ in all the genes analyzed. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII may also play positive roles in other (non-NER) DNA damage repair and/or tolerance mechanisms, as mutations in these regions can cause UV sensitivity that cannot be attributed to derepression of TCR. Our study reveals a novel function of Rpb7 in TCR regulation and suggests that this RNAPII subunit may have broader roles in DNA damage response beyond its known function in transcription.

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.

Genome-wide role of Rad26 in promoting transcription-coupled nucleotide excision repair in yeast chromatin.

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion of SPT4 significantly restores TC-NER across the genome in a rad26∆ mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.

Peptidyl inhibition of Spt4-Spt5: Protein-protein inhibitors for targeting the transcriptional pathway related to C9orf72 expansion repeats.

Spt4/Spt5 is an useful target as it is likely a transcription factor that has implications for long non-coding RNA repeats related to frontotemporal dementia (FTD) found in the C9orf72 disease pathology. Inhibitors for Spt4/Spt5 using peptides as a starting point for assays as a means for developing small molecules, which could likely lead to therapeutic development for inhibition for Spt4/Spt5 with CNS characteristics. To elucidate the specific steps of identification and modification of key interacting residues from Spt4/Spt5 complex with further effect prediction, a set of different computational methods was applied. Newly characterized, theoretically derived peptides docked on Spt4/Spt5 models, based on X-ray crystallography sources, allowed us to complete molecular dynamics simulations and docking studies for peptide libraries that give us high confident set of peptides for use to screen for Spt4/Spt5 inhibition. Several peptides with increased specificity to the Spt4/Spt5 interface were found and can be screened in cell-based assays and enzymatic assays for peptide screens that lead to small molecule campaigns. Spt4/Spt5 comprises an attractive target for neurological diseases, and applying these peptides into a screening campaign will promote the goal of therapeutic searches for FTD drug discovery.

Virulence protein VirD5 of Agrobacterium tumefaciens binds to kinetochores in host cells via an interaction with Spt4.

The bacterium Agrobacterium tumefaciens causes crown gall tumor formation in plants. During infection the bacteria translocate an oncogenic piece of DNA (transferred DNA, T-DNA) into plant cells at the infection site. A number of virulence proteins are cotransported into host cells concomitantly with the T-DNA to effectuate transformation. Using yeast as a model host, we find that one of these proteins, VirD5, localizes to the centromeres/kinetochores in the nucleus of the host cells by its interaction with the conserved protein Spt4. VirD5 promotes chromosomal instability as seen by the high-frequency loss of a minichromosome in yeast. By using both yeast and plant cells with a chromosome that was specifically marked by a lacO repeat, chromosome segregation errors and the appearance of aneuploid cells due to the presence of VirD5 could be visualized in vivo. Thus, VirD5 is a prokaryotic virulence protein that interferes with mitosis.

Identification of Regions in the Spt5 Subunit of DRB Sensitivity-inducing Factor (DSIF) That Are Involved in Promoter-proximal Pausing.

DRB sensitivity-inducing factor (DSIF or Spt4/5) is a conserved transcription elongation factor that both inhibits and stimulates transcription elongation in metazoans. In Drosophila and vertebrates, DSIF together with negative elongation factor (NELF) associates with RNA polymerase II during early elongation and causes RNA polymerase II to pause in the promoter-proximal region of genes. The mechanism of how DSIF establishes pausing is not known. We constructed Spt5 mutant forms of DSIF and tested their capacity to restore promoter-proximal pausing to DSIF-depleted Drosophila nuclear extracts. The C-terminal repeat region of Spt5, which has been implicated in both inhibition and stimulation of elongation, is dispensable for promoter-proximal pausing. A region encompassing KOW4 and KOW5 of Spt5 is essential for pausing, and mutations in KOW5 specifically shift the location of the pause. RNA cross-linking analysis reveals that KOW5 directly contacts the nascent transcript, and deletion of KOW5 disrupts this interaction. Our results suggest that KOW5 is involved in promoter-proximal pausing through contact with the nascent RNA.

Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest.

RNA polymerase II (RNAPII) undergoes structural changes during the transitions from initiation, elongation, and termination, which are aided by a collection of proteins called elongation factors. NusG/Spt5 is the only elongation factor conserved in all domains of life. Although much information exists about the interactions between NusG/Spt5 and RNA polymerase in prokaryotes, little is known about how the binding of eukaryotic Spt4/5 affects the biochemical activities of RNAPII. We characterized the activities of Spt4/5 and interrogated the structural features of Spt5 required for it to interact with elongation complexes, bind nucleic acids, and promote transcription elongation. The eukaryotic specific regions of Spt5 containing the Kyrpides, Ouzounis, Woese domains are involved in stabilizing the association with the RNAPII elongation complex, which also requires the presence of the nascent transcript. Interestingly, we identify a region within the conserved NusG N-terminal (NGN) domain of Spt5 that contacts the non-template strand of DNA both upstream of RNAPII and in the transcription bubble. Mutating charged residues in this region of Spt5 did not prevent Spt4/5 binding to elongation complexes, but abrogated the cross-linking of Spt5 to DNA and the anti-arrest properties of Spt4/5, thus suggesting that contact between Spt5 (NGN) and DNA is required for Spt4/5 to promote elongation. We propose that the mechanism of how Spt5/NGN promotes elongation is fundamentally conserved; however, the eukaryotic specific regions of the protein evolved so that it can serve as a platform for other elongation factors and maintain its association with RNAPII as it navigates genomes packaged into chromatin.

Hot1 factor recruits co-activator Sub1 and elongation complex Spt4/5 to osmostress genes.

Hyperosmotic stress response involves the adaptative mechanisms needed for cell survival. Under high osmolarity conditions, many stress response genes are activated by several unrelated transcription factors that are controlled by the Hog1 kinase. Osmostress transcription factor Hot1 regulates the expression of several genes involved in glycerol biosynthesis, and the presence of this transcription factor in their promoters is essential for RNApol II recruitment. The physical association between Hog1 and Hot1 activates this transcription factor and directs the RNA polymerase II localization at these promoters. We, herein, demonstrate that physical and genetic interactions exist between Hot1 and several proteins involved in transcriptional and posttranscriptional processes: for example, transcription co-activator Sub1 and elongation complex Spt4/5. The results presented in this work demonstrate that Hot1 enrichment is not detected through the coding regions of its target genes and rule out a direct role in transcription elongation. Instead, other data presented herein indicate a key function of the Hot1 transcription factor in the recruitment of these proteins to the promoter or the 5'-coding region of the genes under its control.

Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.

Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5.

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

Spt4 promotes cellular senescence by activating non-coding RNA transcription in ribosomal RNA gene clusters.

Genome instability can drive aging in many organisms. The ribosomal RNA gene (rDNA) cluster is one of the most unstable regions in the genome and the stability of this region impacts replicative lifespan in budding yeast. To understand the underlying mechanism, we search for yeast mutants with stabler rDNA and longer lifespans than wild-type cells. We show that absence of a transcription elongation factor, Spt4, results in increased rDNA stability, reduced levels of non-coding RNA transcripts from the regulatory E-pro promoter in the rDNA, and extended replicative lifespan in a SIR2-dependent manner. Spt4-dependent lifespan restriction is abolished in the absence of non-coding RNA transcription at the E-pro locus. The amount of Spt4 increases and its function becomes more important as cells age. These findings suggest that Spt4 is a promising aging factor that accelerates cellular senescence through rDNA instability driven by non-coding RNA transcription.

Spt4 Promotes Pol I Processivity and Transcription Elongation.

RNA polymerases (Pols) I, II, and III collectively synthesize most of the RNA in a eukaryotic cell. Transcription by Pols I, II, and III is regulated by hundreds of trans-acting factors. One such protein, Spt4, has been previously identified as a transcription factor that influences both Pols I and II. Spt4 forms a complex with Spt5, described as the Spt4/5 complex (or DSIF in mammalian cells). This complex has been shown previously to directly interact with Pol I and potentially affect transcription elongation. The previous literature identified defects in transcription by Pol I when SPT4 was deleted, but the necessary tools to characterize the mechanism of this effect were not available at the time. Here, we use a technique called Native Elongating Transcript Sequencing (NET-seq) to probe for the global occupancy of Pol I in wild-type (WT) and spt4△ Saccharomyces cerevisiae (yeast) cells at single nucleotide resolution in vivo. Analysis of NET-seq data reveals that Spt4 promotes Pol I processivity and enhances transcription elongation through regions of the ribosomal DNA that are particularly G-rich. These data suggest that Spt4/5 may directly affect transcription elongation by Pol I in vivo.

Spt4 facilitates the movement of RNA polymerase II through the +2 nucleosomal barrier.

Spt4 is a transcription elongation factor with homologs in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, and yet the in vivo function of Spt4 is unclear. Here, we assess the precise position of Spt4 during transcription and the consequences of the loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription, and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

CRISPRi-mediated depletion of Spt4 and Spt5 reveals a role for DSIF in the control of HIV latency.

Pivotal studies on the control of HIV transcription has laid the foundations for our understanding of how metazoan transcription is executed, and what are the factors that control this step. Part of this work established a role for DRB Sensitivity Inducing Factor (DSIF), consisting of Spt4 and Spt5, in promoting pause-release of RNA Polymerase II (Pol II) for optimal elongation. However, while there has been substantial progress in understanding the role of DSIF in mediating HIV gene transcription, its involvement in establishing viral latency has not been explored. Moreover, the effects of depleting Spt4 or Spt5, or simultaneously knocking down both subunits of DSIF have not been examined. In this study, we employed CRISPR interference (CRIPSRi) to knockdown the expression of Spt4, Spt5 or the entire DSIF complex, and monitored effects on HIV transcription and viral latency. Knocking down DSIF, or each of its subunits, inhibited HIV transcription, primarily at the step of Tat transactivation. This was accompanied by a decrease in promoter occupancy of Pol II and Cdk9, and to a lesser extent, AFF4. Interestingly, targeting the expression of one subunit of DSIF, reduced the protein stability of its counterpart partner. Moreover, depletion of Spt4, Spt5 or DSIF complex impaired cell growth, but did not cause cell death. Finally, knockdown of Spt4, Spt5 or DSIF, facilitated entry of HIV into latency. We conclude that each DSIF subunit plays a role in maintaining the stability of its other partner, achieving optimal function of the DSIF to enhance viral gene transcription.

Coupling of Transcription and Translation in Archaea: Cues From the Bacterial World.

The lack of a nucleus is the defining cellular feature of bacteria and archaea. Consequently, transcription and translation are occurring in the same compartment, proceed simultaneously and likely in a coupled fashion. Recent cryo-electron microscopy (cryo-EM) and tomography data, also combined with crosslinking-mass spectrometry experiments, have uncovered detailed structural features of the coupling between a transcribing bacterial RNA polymerase (RNAP) and the trailing translating ribosome in Escherichia coli and Mycoplasma pneumoniae. Formation of this supercomplex, called expressome, is mediated by physical interactions between the RNAP-bound transcription elongation factors NusG and/or NusA and the ribosomal proteins including uS10. Based on the structural conservation of the RNAP core enzyme, the ribosome, and the universally conserved elongation factors Spt5 (NusG) and NusA, we discuss requirements and functional implications of transcription-translation coupling in archaea. We furthermore consider additional RNA-mediated and co-transcriptional processes that potentially influence expressome formation in archaea.

The transcription factor Spt4-Spt5 complex regulates the expression of ATG8 and ATG41.

Macroautophagy/autophagy, a highly conserved dynamic process, is one of the major degradative pathways in cells. So far, over 40 autophagy-related (ATG) genes have been identified in Saccharomyces cerevisiae, most of which have homologs in more complex eukaryotes. Autophagy plays a crucial role in cell survival and maintenance, and its dysfunction is related to various diseases, indicating that the proper regulation of autophagy is important. Although the overall process of autophagy has been extensively studied, in particular with regard to the function of the Atg proteins, relatively little is known about the regulatory mechanisms that control autophagy activity. Spt5 is one of the transcriptional factors that is universally conserved across all domains. This protein can form a complex with Spt4, together playing a central role in transcription. In complex eukaryotic cells, the Spt4-Spt5 complex plays a dual role in gene regulation, acting both to delay transcription through promoter-proximal pausing, and to facilitate transcriptional elongation. In contrast, in S. cerevisiae, only the positive function of the Spt4-Spt5 complex has been identified. Here, we show for the first time that the Spt4-Spt5 transcription factor complex negatively regulates ATG genes in S. cerevisiae, inhibiting autophagy activity during active growth. Under autophagy-inducing conditions, the repression is released by Spt5 phosphorylation, allowing an upregulation of autophagy activity. ABBREVIATIONS: AID: auxin-inducible degron; ATG: autophagy-related; ChIP: chromatin immunoprecipitation;Cvt: cytoplasm-to-vacuole targeting; DSIF: DRB sensitivity-inducible factor; NELF: negativeelongation factor; ORF: open reading frame; PA: protein A; PE: phosphatidylethanolamine;prApe1: precursor aminopeptidase I; RT-qPCR: real-time quantitative PCR; RNAP II: RNApolymerase II; TSS: transcription start site; WT: wild-type.

Archaeal transcription.

Increasingly sophisticated biochemical and genetic techniques are unraveling the regulatory factors and mechanisms that control gene expression in the Archaea. While some similarities in regulatory strategies are universal, archaeal-specific regulatory strategies are emerging to complement a complex patchwork of shared archaeal-bacterial and archaeal-eukaryotic regulatory mechanisms employed in the archaeal domain. The prokaryotic archaea encode core transcription components with homology to the eukaryotic transcription apparatus and also share a simplified eukaryotic-like initiation mechanism, but also deploy tactics common to bacterial systems to regulate promoter usage and influence elongation-termination decisions. We review the recently established complete archaeal transcription cycle, highlight recent findings of the archaeal transcription community and detail the expanding post-initiation regulation imposed on archaeal transcription.