We report the cDNA cloning and functional characterization of human cyclin L, a novel cyclin related to the C-type cyclins that are involved in regulation of RNA polymerase II (pol II) transcription. Cyclin L also contains a COOH-terminal dipeptide repeat of alternating arginines and serines, a hallmark of the SR family of splicing factors. We show that recombinant cyclin L interacts with p110 PITSLRE kinase, and that cyclin L antibody co-immunoprecipitates a kinase activity from HeLa nuclear extracts that phosphorylates the carboxyl-terminal domain (CTD) of pol II and splicing factor SC35, and is inhibited by the cdk inhibitor p21. Cyclin L antibody inhibits the second step of RNA splicing in vitro, and recombinant cyclin L protein stimulates splicing under suboptimal conditions. Significantly, the IC 50 for splicing inhibition by p21 is similar to the IC 50 for inhibition of the cyclin L-associated kinase activity. Cyclin L and its associated kinase are thus new members of the pre-mRNA processing machinery.Cyclins and their partners the cyclin-dependent kinases (cdks) 1 (reviewed in Refs. 1 and 2) may be classified into two major groups according to their function: the cell cycle regulators, which include the cyclin classes A, B, D, and E and cdks 1, 2, 3, 4, and the transcriptional regulators, comprising the cyclin classes C, H, K, and T and cdks 7, 8, and 9. These latter cyclin/kinase pairs are associated with the transcriptional machinery, and are components of transcription factor TFIIH (3-7), elongation factor P-TEFb, (8 -10), and the RNA polymerase II holoenzyme (11-13). Human cyclin K is homologous to cyclin C, associates with the large subunit of pol II, and its kinase partner, cdk9, phosphorylates the CTD of pol II (14).Phosphorylation of the CTD plays a pivotal role in regulating transcription initiation, elongation, and processing of RNA transcripts. It is widely accepted that transcription and RNA processing are linked (reviewed in Ref. 15): capping enzymes, polyadenylation factors, and splicing factors assemble at the CTD, and these interactions are modulated by CTD phosphorylation (reviewed in Refs. 16 -18). Extensive research has focused on the role of the CTD in regulating pre-mRNA splicing. The CTD targets splicing factors to transcription sites in vivo (19); phosphorylated pol II stimulates splicing (20), and splicing factors associate with pol II through a hyperphosphorylated CTD (21).Splicing factors comprise the small nuclear ribonucleoprotein particles, the spliceosome-associated proteins, and the SR proteins (reviewed in Ref. 22). SR proteins constitute a conserved family of pre-mRNA splicing factors that are characterized by an arginine-serine dipeptide repeat within their carboxyl-terminal domain and one or two RNA-binding domains within their amino-terminal domain (23). SR proteins are essential splicing factors, and are capable of complementing splicing-deficient cellular extracts. Several members of this family have been identified, and among these the human factors ASF/SF2 (24, 25)...
Pyrrole-imidazole (Py-Im) polyamides are synthetic ligands that can be designed to bind predetermined DNA sequences (40,44). These minor-groove DNA-binding molecules block eukaryotic transcription factors from binding to their cognate DNA sequences and inhibit transcription, both in vitro and, in a few cases, in cell culture experiments (reviewed in reference 13). Polyamides are effective inhibitors of tissue-specific and general transcription factors (7, 8) as well as viral repressors (9) and transactivators (29). Recently, activation of gene expression has been achieved in vitro by tethering a small peptide activation domain to a sequence-specific Py-Im polyamide (31). Remarkably, activation and repression of selected genes have been achieved in Drosophila melanogaster by targeting polyamides to highly repeated satellite DNA sequences (17,18).Since batteries of genes utilize common general and tissuespecific transcription factors, polyamides have been synthesized to bind sequences adjacent to the binding sites for required transcription factors (7). A polyamide targeted to sequences adjacent to the human immunodeficiency virus type 1 (HIV-1) TATA box effectively inhibits TATA box binding protein (TBP) binding and basal transcription by RNA polymerase II (7). The binding of the TBP subunit of TFIID in the minor groove nucleates assembly of the polymerase II transcription machinery for TATA-containing genes (24,25). Since TFIID and the other general transcription factors TFIIA, -B, -E, -F, and -H (28, 33) occupy at least 40 bp of promoter DNA upstream from the transcription start site of mRNA-coding genes, this raises the question of whether sites nonoverlapping and distant from the TATA box might also serve as effective polyamide targets for inhibition of transcription. To address this issue, we generated a series of DNA constructs in which a common polyamide-binding site was scanned through a promoter and determined the effect of binding site position on inhibition of TBP binding and basal RNA polymerase II transcription. Our results show that essential protein-DNA contacts on the HIV-1 core promoter are not simply restricted to the TATA box and initiator element (20, 45) but rather extend both upstream and downstream of the TATA box. Some of these contacts are likely due to TFIID, the multiprotein complex containing TBP. Importantly, transcription inhibition can be achieved by targeting polyamides to promoter sequences distant from the TATA element that are gene specific. Py-Im polyamides thus provide simple and convenient chemical probes for discovery of functionally important protein-DNA contacts within specific gene promoters. MATERIALS AND METHODSPolyamide synthesis and characterization. Three Py-Im polyamides (1-3), whose structures are shown in Fig. 1A, were synthesized by solid-phase methods (1). Polyamide-EDTA conjugates were also prepared (40). The purity and identity of each compound were verified by analytical high-pressure liquid chromatography, 1 H nuclear magnetic resonance, and matrix-assisted ...
Human estrogen-related receptor 2 (hERR2, ESRRB, ERRbeta, NR3B2) belongs to a class of nuclear receptors that bind DNA through sequence-specific interactions with a 5'-AGGTCA-3' estrogen response element (ERE) half-site in the major groove and an upstream 5'-TNA-3' site in the minor groove. This minor groove interaction is mediated by a C-terminal extension (CTE) of the DNA binding domain and is unique to the estrogen-related receptors. We have used synthetic pyrrole-imidazole polyamides, which bind specific sequences in the minor groove, to demonstrate that DNA binding by hERR2 is sensitive to the presence of polyamides in both the upstream minor groove CTE site and the minor groove of the ERE half-site. Thus, polyamides can inhibit hERR2 by two mechanisms, by direct steric blockage of minor groove DNA contacts mediated by the CTE and by changing the helical geometry of DNA such that major groove interactions are weakened. To confirm the generality of the latter approach, we show that the dimeric human estrogen receptor alpha (hERalpha, ESR1, NR3A1), which binds in the major groove of the ERE, can be inhibited by a polyamide bound in the opposing minor groove of the ERE. These results highlight two mechanisms for inhibition of protein-DNA interactions and extend the repertoire of DNA recognition motifs that can be inhibited by polyamides. These molecules may thus be useful for controlling expression of hERR2- or hERalpha-responsive genes.
Protein-DNA interactions that lie outside of the core recognition sequence for the Drosophila bHLH transcription factor Deadpan (Dpn) were investigated using minor groove binding pyrrole-imidazole polyamides. Electrophoretic mobility shift assays and DNase I footprinting demonstrate that hairpin polyamides bound immediately upstream, but not immediately downstream of the Dpn homodimer selectively inhibit protein-DNA complex formation. Mutation of the Dpn consensus binding site from the asymmetric sequence 5'-CACGCG-3' to the palindromic sequence 5'-CACGTG-3' abolishes asymmetric inhibition. A Dpn mutant containing the unnatural amino acid norleucine in place of lysine at position 80 in the bHLH loop region is not inhibited by the polyamide, suggesting that the epsilon amino group at this position is responsible for DNA contacts outside the major groove. We conclude that the nonpalindromic Dpn recognition site imparts binding asymmetry by providing unique contacts to the basic region of each monomer in the bHLH homodimer.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.