View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ZENODO Biohelikon: Cell Biology(ISSN: 2348-3741), 2014 2:a18 Review The Negative Elongation Factor Complex – a poorly understood multi- faceted transcript-processing complex Byron Baron1,2* 1Department of Anatomy and Cell Biology, Faculty of Medicine and Surgery, University of Malta, Msida, Malta. 2Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Japan. * Corresponding author, Email: [email protected] Abstract With the advent of whole genome screens, promoter-proximal pausing of RNA polymerase II (RNAPII) has been shown to play a much more significant role in eukaryotic systems than previously thought. This type of transcription inhibition is dependent on the binding of the Negative Elongation Factor (NELF) complex which is composed of four sub-units, presenting unique domains and consequently fulfilling different functions. Numerous questions still surround the mechanism by which NELF is recruited, stabilised and dissociated from the RNAPII complex. Furthermore, not much is known about which other transcription stages the NELF complex is involved in and through which sub-units it carries out such functions. Based on the current knowledge of the role of NELF in transcription pausing, it is hypothesised that different interaction partners are required to direct context-specific functions of the NELF complex. This review covers some of the known roles and contexts in which NELF acts in an attempt to identify key questions for future NELF-dependent transcriptional research. Keywords Transcription inhibition, Negative Elongation Factor complex, promoter-proximal pausing, transcription regulation model, transcription factors Highlights segment of NELF-A shows sequence similarity to the hepatitis delta antigen (HDAg), which binds to RNAPII 1 .The Negative Elongation Factor (NELF) is a multi- and activates transcription elongation, indicating subunit transcription elongation factor that NELF-A mediates the interaction between NELF 2 .The great majority of human protein coding and RNAPII [3,7]. genes exhibit promoter-proximal pausing of RNA NELF-B (also known as cofactor of BRCA1 – Polymerase II (RNAPII) as a result of NELF action COBRA) has been reported to physically interact with 3 .NELF can only bind to a pre-formed 5,6-Dichloro-1- the product of breast/ovarian cancer susceptibility β-D-ribofuranosylbenzimidazole (DRB) Sensitivity- gene, BRCA1 and computer analysis has shown Inducing Factor (DSIF)/RNAPII complex that this interaction occurs between a coiled-coil 4 .NELF is involved in multiple mRNA processing structure in NELF-B and a leucine zipper motif in steps during transcription by interacting with NELF-E [1,11]. It has been shown that NELF-B inhibits different factors the growth of oestrogen receptor α (ERα) positive 5 .Transcription factors e.g. GAGA factor can recruit breast cancer cells in vitro [12] and that NELF-B proteins involved in promoter-proximal pausing of expression is reduced in several established breast RNAPII cancer cell lines [13]. A study of a cohort of breast The NELF complex ductal carcinoma samples from patients with known The Negative Elongation Factor (NELF) complex, clinical outcomes revealed that a low level of NELF- originally isolated through its suppression of B mRNA is associated with metastatic breast cancer transcription elongation, is a multi-subunit [14]. In a different context, mice homozygous for a transcription elongation factor composed of four mutation in NELF-B have an inner cell mass deficiency sub-units: NELF-A (66kDa), NELF-B (62kDa), NELF-C and die at the time of implantation, providing (60kDa) or NELF-D (59kDa), and NELF-E (46kDa) [1-4]. evidence as to the critical role of NELF-B in early NELF induces a process called promoter-proximal mouse embryogenesis [15]. pausing of RNA polymerase II (RNAPII) [5-7]. NELF-C and NELF-D are the least studied of NELF-A is encoded by the WHSC2 gene, a the NELF sub-units. It is known that they arise candidate gene for Wolf-Hirschhorn syndrome – from a common mRNA species through the use of characterised by a distinctive craniofacial phenotype, alternative start codons [1] and that they are highly growth and mental retardation, seizures and cardiac similar in sequence to the Drosophila protein TH1, abnormalities [3,8-10]. Interestingly, the N-terminal which as yet has no known function. However no 1 other functional information has been reported in the literature. The structure of NELF-E is characterised by an N-terminal leucine zipper motif, a central domain rich in arginine and aspartic acid (Arg-Asp or R- D) dipeptide repeats (the RD motif), and a C- terminal RNA recognition motif (RRM). RRMs, which typically encompass 80 to 90 amino acids with two highly conserved elements called RNP1 and RNP2, often bind to RNA in a sequence- or structure- specific manner [16,17]. The RRM of NELF-E is capable of binding to various nascent RNA transcripts synthesised by RNAPII, and in so doing, repressing transcription [4]. Promoter-Proximal Pausing In yeast, RNAPII is typically distributed roughly uniformly across transcription units, suggesting a context in which RNAPII is not regulated after transcription is initiated [18]. However, in higher eukaryotes, RNAPII-binding is concentrated near the Figure 1 Initiation – The transcription factor complex (TF Complex) Transcription Start Site (TSS) of many genes, implying binds to the promoter, recruiting RNA Polymerase II (RNAPII) to the transcription start site (TSS). DSIF, composed of two sub-units (Spt4 the action of a control process referred to as and Spt5), then binds to RNAPII and a short RNA strand begins to form. promoter-proximal pausing. Pausing – The negative elongation factor complex made up of the sub- Promoter-proximal pausing of RNAPII is a units Nelf-A, Nelf-B, Nelf-C/D, Nelf-E (A, B, C/D, E) is recruited to the TSS and hypothesised to be stabilised by binding to the TF complex. The mechanism that finely controls the levels of RNA strand formed binds to the RMM domain of Nelf-E, blocking the gene expression, as opposed to, switching gene elongation process. Elongation – A stimulus brings about recruitment expression on and off. This is brought about of P-TEFb (CyclinT and Cdk9), which phosphorylates RNAPII and DSIF causing the dissociation of the negative elongation factor complex (A, by the physically binding of 5,6-Dichloro-1- B, C/D, E) and allowing the extension of the RNA strand, forming a full β-D-ribofuranosylbenzimidazole (DRB) Sensitivity- transcript. Inducing Factor (DSIF) to RNAPII, without affecting the catalytic activity of the latter, but creating a Biological significance of NELF scaffold for NELF to bind to this pre-formed RNAPII/ DSIF complex [2,4,7]. Transcription inhibition of The importance of NELF and promoter-proximal RNAPII is alleviated by the recruitment of chromatin- pausing of RNAPII is evident in response to specific transcription elongation factor Facilitates intracellular and extracellular stimuli e.g. heat shock, Chromatin Transcription Complex (FACT), together lipopolysaccharide (LPS) and oestrogen [31,37] NELF with the protein kinase Positive Transcription localises with RNAPII just downstream of the TSS of Elongation Factor b (P-TEFb), which through its heat shock genes when they are not induced, where Cyclin-dependent kinase 9 (Cdk9) sub-unit binds to it collaborates with DSIF to establish promoter- and phosphorylates the C-terminal repeat domain proximal pausing of RNAPII, by associating with (CTD) of RNAPII and DSIF. Upon phosphorylation, nascent RNAs, 20–60 nucleotide long [6,38,39]. Upon DSIF is converted from a negative to a positive heat shock, activating factors trigger the release regulator of transcription, NELF is removed of RNAPII from its paused state, and there is a and RNAPII can proceed to produce full-length rapid production of full-length transcripts from Heat transcripts (Fig. 1) [2,7,20-25]. Shock Protein (HSP) genes[6,38,39]. A loss of NELF In Drosophila, promoter-proximal pausing of however, does not affect the rate of heat shock RNAPII is present in approximately 10% of genes gene induction but instead delays the time taken for in early embryos and 20% of genes in adult S2 HSP transcription to decrease down to basal levels cells [26,27], acting as a checkpoint to regulate after the heat shock stimulus has ceased, implying transcription [28,29]. Even more so, in human that the presence of NELF somehow facilitates the embryonic stem cells, approximately 75% of protein dissociation of the transcription factor Heat Shock coding genes exhibit promoter-proximal pausing of Factor (HSF) [40]. RNAPII, while only about half of these genes produce NELF activity is also important during detectable full-length transcripts [30]. development e.g. in early Drosophila development, mutant clones lacking NELF-A or NELF-E either stopped developing before the blastoderm stage (displaying abnormal nuclear morphology) or during germ-band retraction (exhibiting head defects and incomplete dorsal closure). However, it appears that abnormal NELF has no effect on segmentation genes (e.g. eve, fushi tarazu, and sloppy paired 1) at the blastoderm stage [41]. NELF has also been shown to 2 play a critical role in the neuronal development of paused RNAPII is only found is a sub-set of these zebra fish embryos [42-44]. genes, indicating that NELF does not only fulfil the Other contexts involving NELF regulation include role of repressor of gene expression [60]. This is the attenuation of transcription levels of oestrogen- achieved through interaction with different factors. responsive genes following stimulation [12], the One protein which displays a similar functional- attenuation of transcription levels of the immediate- flexibility to NELF is the nuclear cap binding complex early genes and the transcription factor junB, both (CBC), which was originally found to bind to the 5’ cap before and after induction [5], as well as the structure of maturing RNA and facilitates its export activation of paused genes involved in the immune [61].