Negative elongation factor (NELF) is a four subunit transcription elongation factor that has been implicated in numerous diseases ranging from neurological disorders to cancer. Here we show that NELF interacts with the nuclear cap binding complex (CBC), a multifunctional factor that plays important roles in several mRNA processing steps, and the two factors together participate in the 3' end processing of replication-dependent histone mRNAs, most likely through association with the histone stem-loop binding protein (SLBP). Strikingly, absence of NELF and CBC causes aberrant production of polyadenylated histone mRNAs. Moreover, NELF is physically associated with histone gene loci and forms distinct intranuclear foci that we call NELF bodies, which often overlap with Cajal bodies and cleavage bodies. Our results point to a surprising role of NELF in the 3' end processing of histone mRNAs and also suggest that NELF is a new factor that coordinates different mRNA processing steps during transcription.

The elongation step of RNA polymerase II (RNAPII) transcription is emerging as a critical control point for the expression of various genes and for diverse biological processes. Examples include neuronal fate determination during embryonic development (6, 44), gene expression of human immunodeficiency virus (5,11,13,19,43), replication and transcription of hepatitis delta virus (38), and transcriptional regulation of heat shock genes (1,10,18). In all these cases, the involvement of three transcription elongation factors, namely, DRB (5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF), NELF (negative elongation factor), and positive transcription elongation factor b (P-TEFb), has been demonstrated or implicated.Shortly after the initiation of transcription, RNAPII comes under the negative and positive control of DSIF, NELF, and P-TEFb. DSIF and NELF cause transcriptional pausing through physical association with RNAPII. DSIF binds to RNAPII directly and stably (33, 36). However, this appears to have little effect on the catalytic activity of RNAPII (37). A previous study has pointed out that NELF does not bind substantially to DSIF or RNAPII alone but does bind to the complex of DSIF and RNAPII (40). This association is the likely trigger of transcriptional pausing. Conversely, P-TEFb allows RNAPII to enter the productive elongation phase by preventing the action of DSIF and NELF (27, 37). P-TEFb is the protein kinase whose primary target is thought to be the C-terminal domain (CTD) of RNAPII (26). Most, but not all, evidence suggests that P-TEFb-dependent phosphorylation of the CTD facilitates the release of DSIF and NELF from RNA-PII, thereby reversing the inhibition (3, 24, 37). In theory, such regulation at the elongation step allows for rapid change in mRNA levels and for highly sophisticated control over gene expression when combined with regulation at the (pre)initiation step.The structures and functions of DSIF and P-TEFb have been extensively characterized. Human DSIF is a heterodimer composed of p14 (14 kDa) and p160 (160 kDa), whose Saccharomyces cerevisiae counterparts are Spt4 and Spt5 (7,33). In addition to its role in transcriptional pausing, DSIF has a potential to activate RNAPII elongation. The activation mechanism is not well understood: interaction partners of DSIF other than NELF may be involved (13,14,20,23,28). Spt5 has a highly acidic N-terminal region, multiple copies of the KOW motifs, and a repetitive C-terminal region analogous to the RNAPII CTD (9,25,36). RNAPII interacts with Spt5 through a region encompassing the KOW motifs. KOW motifs are also found in the bacterial transcription elongation factor NusG, which binds to prokaryotic RNA polymerase and controls termination and antitermination (15,17,29). In addition, the extreme C terminus of Spt5 is specifically involved in the transcriptional repression pathway (6). Human P-TEFb is a heterodimer composed of Cdk9 (41 kDa) and one of multiple cyclin subunits T1, T2a, T2b, and K (50 to 90 kDa) (26). The k...

Negative elongation factor (NELF) is a human transcription factor complex that cooperates with DRB sensitivity-inducing factor (DSIF)/hSpt4-hSpt5 to repress elongation by RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, including NELF-A, a candidate gene product for Wolf-Hirschhorn syndrome, and NELF-E, a putative RNA-binding protein with arginine-aspartic acid (RD) dipeptide repeats. Here we report several important findings regarding the DSIF/NELF-dependent elongation control. First, we have established an effective method for purifying the active NELF complex using an epitope-tagging technique. Second, the five polypeptides each are important and together are sufficient for its function in vitro. Third, NELF does not bind to either DSIF or RNAPII alone but does bind to the preformed DSIF/RNAPII complex. Fourth, NELF-E has a functional RNA-binding domain, whose mutations impair transcription repression without affecting known protein-protein interactions. Taken together, we propose that NELF causes RNAPII pausing through binding to the DSIF/RNAPII complex and to nascent transcripts. These results also have implications for how DSIF and NELF are regulated in a gene-specific manner in vivo.Transcription elongation by RNA polymerase II (RNAPII) is controlled by a number of trans-acting factors called transcription elongation factors as well as by cis-acting elements (2, 26). Transcription elongation factors such as transcription factor IIF (TFIIF), elongin, and TFIIS interact with elongating RNAPII to prevent its pausing or to reactivate it from an arrested configuration. cis-acting elements are mainly located on nascent transcripts. Some RNA elements cause RNAPII to pause or arrest without the aid of protein factors by forming structures that destabilize RNAPII-DNA-RNA complexes (19,26). Other types of RNA elements include the one called TAR, which exists at the 5Ј end of human immunodeficiency virus (HIV) transcripts and serves as a binding site for the viral activator Tat and cellular cofactors (9); together these strongly stimulate RNAPII elongation. The functions of many other RNA elements are largely unknown.The recent discovery of a new class of positive and negative elongation factors, including DRB sensitivity-inducing factor (DSIF), negative elongation factor (NELF), and positive transcription elongation factor b (P-TEFb), has shed new light on the control of RNAPII elongation (21,27,(31)(32)(33)35). Biochemical studies have established that DSIF and NELF cooperatively repress RNAPII elongation, whereas P-TEFb alleviates the repression in a manner sensitive to the kinase inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) (28,29,31). DSIF and NELF coimmunoprecipitate with the unphosphorylated form of RNAPII (IIa), but not with the hyperphosphorylated form (IIo) (28, 31). In addition, P-TEFb strongly phosphorylates the C-terminal domain (CTD) of RNAPII and a subunit of DSIF in a DRB-sensitive manner (8,12,35). From these data, we have proposed that transcription r...

Xeroderma pigmentosum group D (XPD) protein is one of the subunits of TFIIH that is required for nucleotide excision repair and transcription. We found a XPD protein complex containing MMS19 that was assumed to be a regulator of TFIIH. However, the MMS19-XPD complex did not contain any other subunits of TFIIH. Instead, it included FAM96B (now designated MIP18), Ciao1, and ANT2. MMS19, MIP18, and XPD localized to the mitotic spindle during mitosis. The siRNA-mediated knockdown of MMS19, MIP18, or XPD led to improper chromosome segregation and the accumulation of nuclei with abnormal shapes. In addition, the frequency of abnormal mitosis and nuclei was increased in XP-D and XP-D/CS patients' cells. These results indicate that the MMS19-XPD protein complex, now designated MMXD (MMS19-MIP18-XPD), is required for proper chromosome segregation, an abnormality of which could contribute to the pathogenesis in some cases of XP-D and XP-D/CS.

Transcription elongation by RNA polymerase II (RNAPII) is negatively regulated by the human factors DRB-sensitivity inducing factor (DSIF) and negative elongation factor (NELF). A 66-kilodalton subunit of NELF (NELF-A) shows limited sequence similarity to hepatitis delta antigen (HDAg), the viral protein required for replication of hepatitis delta virus (HDV). The host RNAPII has been implicated in HDV replication, but the detailed mechanism and the role of HDAg in this process are not understood. We show that HDAg binds RNAPII directly and stimulates transcription by displacing NELF and promoting RNAPII elongation. These results suggest that HDAg may regulate RNAPII elongation during both cellular messenger RNA synthesis and HDV RNA replication.