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A wide range of sequencing methods has been developed to assess nascent RNA transcription and resolve the single-nucleotide position of RNA polymerase genome-wide. These techniques are often burdened with high input material requirements and lengthy protocols. We leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT) and developed Butt-seq (bulk analysis of nascent transcript termini sequencing), which can produce libraries from purified nascent RNA in 6 h and from as few as 10,000 cells-an improvement of at least 10-fold over existing techniques. Butt-seq shows that inhibition of the superelongation complex (SEC) causes promoter-proximal pausing to move upstream in a fashion correlated with subnucleosomal fragments. To address transcriptional regulation in a tissue, Butt-seq was used to measure the circadian regulation of transcription from fly heads. All the results indicate that Butt-seq is a simple and powerful technique to analyze transcription at a high level of resolution., (© 2023 Yu and Rosbash; Published by Cold Spring Harbor Laboratory Press.)

The mammalian circadian clock relies on the master genes CLOCK and BMAL1 to drive rhythmic gene expression and regulate biological functions under circadian control. Here we show that rhythmic CLOCK:BMAL1 DNA binding promotes rhythmic chromatin opening. Mechanisms include CLOCK:BMAL1 binding to nucleosomes and rhythmic chromatin modification; e.g., incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLOCK:BMAL1, suggesting that the activity of these other transcription factors contributes to the genome-wide CLOCK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation.

A conserved transcriptional feedback loop underlies animal circadian rhythms. In Drosophila, the transcription factors CLOCK (CLK) and CYCLE (CYC) activate the transcription of direct target genes like period (per) and timeless (tim). They encode the proteins PER and TIM, respectively, which repress CLK/CYC activity. Previous work indicates that repression is due to a direct PER-CLK/CYC interaction as well as CLK/CYC phosphorylation. We describe here the role of ubiquitin-specific protease 8 (USP8) in circadian transcriptional repression as well as the importance of CLK ubiquitylation in CLK/CYC transcription activity. usp8 loss of function (RNAi) or expression of a dominant-negative form of the protein (USP8-DN) enhances CLK/CYC transcriptional activity and alters fly locomotor activity rhythms. Clock protein and mRNA molecular oscillations are virtually absent within circadian neurons of USP8-DN flies. Furthermore, CLK ubiquitylation cycles robustly in wild-type flies and peaks coincident with maximal CLK/CYC transcription. As USP8 interacts with CLK and expression of USP8-DN increases CLK ubiquitylation, the data indicate that USP8 deubiquitylates CLK, which down-regulates CLK/CYC transcriptional activity. Taken together with the facts that usp8 mRNA cycles and that its transcription is activated directly by CLK/CYC, USP8, like PER and TIM, contributes to the transcriptional feedback loop cycle that underlies circadian rhythms.

To determine the prevalence of cotranscriptional splicing in Drosophila, we sequenced nascent RNA transcripts from Drosophila S2 cells as well as from Drosophila heads. Eighty-seven percent of the introns assayed manifest >50% cotranscriptional splicing. The remaining 13% are cotranscriptionally spliced poorly or slowly, with ∼3% being almost completely retained in nascent pre-mRNA. Although individual introns showed slight but statistically significant differences in splicing efficiency, similar global levels of splicing were seen from both sources. Importantly, introns with low cotranscriptional splicing efficiencies are present in the same primary transcript with efficiently spliced introns, indicating that splicing is intron-specific. The analysis also indicates that cotranscriptional splicing is less efficient for first introns, longer introns, and introns annotated as alternative. Finally, S2 cells expressing the slow RpII215(C4) mutant show substantially less intron retention than wild-type S2 cells.

CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4-6 h later by the transcriptional repressor PERIOD (PER), indicating that the majority of CLK targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation.

Transcriptional feedback loops are central to the generation and maintenance of circadian rhythms. In animal systems as well as Neurospora, transcriptional repression is believed to occur by catalytic post-translational events. We report here in the Drosophila model two different mechanisms by which the circadian repressor PERIOD (PER) inhibits CLOCK/CYCLE (CLK/CYC)-mediated transcription. First, PER is recruited to circadian promoters, which leads to the nighttime decrease of CLK/CYC activity. This decrease is proportional to PER levels on DNA, and PER recruitment probably occurs via CLK. Then CLK is released from DNA and sequestered in a strong, approximately 1:1 PER-CLK off-DNA complex. The data indicate that the PER levels bound to CLK change dynamically and are important for repression, first on-DNA and then off-DNA. They also suggest that these mechanisms occur upstream of post-translational events, and that elements of this two-step mechanism likely apply to mammals.

Little is known about the contribution of translational control to circadian rhythms. To address this issue and in particular translational control by microRNAs (miRNAs), we knocked down the miRNA biogenesis pathway in Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify miRNA-mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs-clock (clk), vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, we exploited circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling array analysis. This approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell biochemical experiments indicate that bantam regulates the translation of clk through an association with three target sites located within the clk 3' untranslated region (UTR). Moreover, clk transgenes harboring mutated bantam sites in their 3' UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes.

Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, PER and TIM, repress CLK-CYC-mediated transcription. To recognize additional direct CLK target genes, we designed a genome-wide approach and identified clockwork orange (cwo) as a new core clock component. cwo encodes a transcriptional repressor that synergizes with PER and inhibits CLK-mediated activation. Consistent with this function, the mRNA profiles of CLK direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that CWO acts preferentially in the late night to help terminate CLK-CYC-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks.

Spliceosome assembly in the budding yeast Saccharomyces cerevisiae was recently shown to occur at the site of transcription. However, evidence for cotranscriptional splicing as well as for coupling between transcription and splicing is still lacking. Using modifications of a previously published chromatin immunoprecipitation (ChIP) assay, we show that cotranscriptional splicing occurs approximately 1 kb after transcription of the 3' splice site (3'SS). This pathway furthermore protects most intron-containing nascent transcripts from the effects of cleavage by an intronic hammerhead ribozyme. This suggests that a high percentage of introns are recognized cotranscriptionally. This observation led us to screen a small deletion library for strains that sensitize a splicing reporter to ribozyme cleavage. Characterization of the Deltamud2 strain indicates that the early splicing factor Mud2p functions with U1 snRNP to form a cross-intron bridging complex on nascent pre-mRNA. The complex helps protect the transcript from ribozyme-mediated destruction and suggests an intron-definition event early in the spliceosome assembly process. The transcription elongation mutant strains Deltadst1 and Deltapaf1 show different cotranscriptional splicing phenotypes, suggesting that different transcription pathways differentially impact the efficiency of nascent intron definition.

In the yeast commitment complex and the mammalian E complex, there is an important base-pairing interaction between the 5' end of U1 snRNA and the conserved 5' splice site region of pre-mRNA. But no protein contacts between splicing proteins and the pre-mRNA substrate have been defined in or near this region of early splicing complexes. To address this issue, we used 4-thiouridine-substituted 5' splice site-containing RNAs as substrates and identified eight cross-linked proteins, all of which were identified previously as commitment complex components. The proteins were localized to three domains: the exon, the six nucleotides of the 5' ss region, and the downstream intron. The results indicate that the 5' splice site region and environs are dense with protein contacts in the commitment complex and suggest that some of them make important contributions to formation or stability of the U1 snRNP-pre-mRNA complex.

During the early events of pre-mRNA splicing, intronic cis-acting sequences are recognized and interact through a network of RNA-RNA, RNA-protein, and protein-protein contacts. Recently, we identified a branchpoint sequence binding protein in yeast (BBP). The mammalian ortholog (mBBP/SF1) also binds specifically to branchpoint sequences and interacts with the well studied mammalian splicing factor U2AF65, which binds to the adjacent polypyrimidine (PY) tract. In this paper we demonstrate that the mBBP/SF1-U2AF65 interaction promotes cooperative binding to a branchpoint sequence-polypyrimidine tract-containing RNA, and we suggest that this cooperative RNA binding contributes to initial recognition of the branchpoint sequence (BPS) during pre-mRNA splicing. We also demonstrate the essential nature of the third RBD of U2AF65 for the interaction between the two proteins, both in the presence and absence of RNA.

The FG-repeat domain of the yeast Rip1 protein (Rip1p) was identified initially as a possible target for the nuclear export signal (NES) of the HIV-1 Rev protein in a yeast two-hybrid assay. Rip1p is inessential, associated with nuclear pore complexes, and structurally related to the FG-nucleoporin family of pore proteins. It contributes to HIV-1 Rev-mediated RNA export and is also important for the export of heat shock RNAs at 42 degrees C. We show here that Rip1p is essential for the export of heat shock RNAs, and this function is fulfilled by the unique carboxyl terminus of Rip1p with no substantial contribution from the FG-repeat region. Genetic interactions between Rip1p and the RNA export mediator Gle1p are described, which support a role of the carboxyl terminus of Rip1p in poly(A)+ RNA export. Finally, this domain of Rip1p also contributes to Rev-mediated RNA export. The data suggest that Rip1p promotes the nuclear export of different classes of substrates by contributing to optimal pore function.

The mechanism by which pre-mRNAs are initially recognized by the splicing machinery is not well understood. In the yeast system, commitment complexes are the earliest identified splicing complexes. They contain pre-mRNA, U1 snRNP, and the splicing factor Mud2p and probably correspond to the mammalian E complexes, which contain pre-mRNA, U1 snRNP, and the splicing factor U2AF. To identify other yeast commitment complex components, we have characterized mutant strains that are synthetic lethal with viable U1 snRNA mutations. We report here that MUD13 is a nonessential gene that encodes the yeast homolog of CBP20, the small subunit of the vertebrate nuclear cap-binding complex (CBC). Characterization of splicing in the delta-MUD13 strain and extract indicates that Mud13p is a yeast splicing factor and is the second identified non-snRNP commitment complex component. The observations also suggest that CBC interacts with other commitment complex components as well as with the substrate cap. Taken together with the accompanying results for a mammalian system, our data indicate that cap-binding proteins as well as the pre-mRNA cap contribute to early steps in spliceosome assembly.

In characterizing a series of yeast (Saccharomyces cerevisiae) mutants synthetic lethal with U1 RNA, we have identified a yeast gene (MUD2) with sequence similarity to the well-studied metazoan splicing factor U2AF65. The biochemical characterization indicates that the MUD2 gene product (MUD2P) contacts pre-mRNA directly and is a component of the pre-mRNA-U1 snRNP complex (commitment complex) that forms during early spliceosome assembly in yeast extracts. Unlike U1 snRNP itself, the association of MUD2P with pre-mRNA is dependent on a proper yeast branchpoint sequence. Genetic experiments indicate that MUD2P affects U2 snRNP addition. Moreover, experiments in the two-hybrid system show that PRP11P, a recently identified component of U2 snRNP, can interact directly with MUD2P. The experiments identify a specific inter-snRNP protein-protein contact that occurs during spliceosome assembly and more generally support substantial functional similarity between U2AF65 and MUD2P.

In an enhancer screen for yeast mutants that may interact with U1 small nuclear RNA (snRNA), we identified a gene that encodes the apparent yeast homolog of the well-studied human U1A protein. Both in vitro and in vivo, the absence of the protein has a dramatic effect on the activity of U1 snRNP containing the mutant U1 snRNA used in the screen. Surprisingly, the U1A gene is inessential in a wild-type U1 RNA background, as growth rate and the splicing of endogenous pre-mRNA transcripts are normal in these strains that lack the U1A protein. Even in vitro, the absence of the protein has little effect on splicing. On the basis of these observations, we suggest that a principal role of the U1A protein is to help fold or maintain U1 RNA in an active configuration.

A temperature-sensitive lethal mutation in Saccharomyces cerevisiae, prp20-1, causes defects in several different steps in mRNA metabolism, including mRNA 3'-end formation, transcription initiation, and mRNA transport. Previous work has demonstrated that prp20 mutants are defective in actin pre-mRNA splicing. PRP20 is related, both in structure and function, to the RCC1 gene of mammals and the PIM1 gene of Schizosaccharomyces pombe, both of which appear to regulate entry into mitosis and chromosome condensation. In this report we demonstrate that, after a shift of prp20 mutants to the restrictive temperature, transcripts of several genes (CUP1, CYH2, and GAL10) are produced that extend 1-10 kb beyond their normal polyadenylation sites. The failure in 3'-end formation occurs within 1-2 min of the temperature shift. Transcription initiation also is disrupted, in that initiation sites upstream of the normal cap site are used. mRNA transport from nucleus to cytoplasm also is perturbed: In situ hybridization using an oligo(dT) probe demonstrates accumulation of poly(A) in the nucleus, consistent with the accumulation of longer bulk poly(A) (up to approximately 90-100 nucleotides) and with a failure to transport newly synthesized RNA to the cytoplasm. We demonstrate that prp20 and rna1 mutants are very similar, if not identical, with respect to each of these biochemical phenotypes. In light of the putative role of PRP20 in mitotic control, our results suggest a common step in that process and multiple steps in mRNA synthesis and maturation. We speculate that the perturbations in mRNA processing are the result of effects on the chromatin-nascent RNP-transcription complex or misregulation of a cell cycle component that modifies multiple mRNA-processing activities.

To address the mechanisms that underlie splice site selection and splice site partner assignment, we analyzed the splicing of yeast (Saccharomyces cerevisiae) transcripts containing splice site region duplications. When the 5'-splice site region was duplicated, both sites were utilized to the same extent, indicating little or no influence of proximity on 5'-splice site choice. However, the effect of a 5'-mutant site was greatly enhanced by the presence of an adjacent wild-type site, and this effect was reversed by the restoration of base-pairing with U1 snRNA. 3'-Splice site choice was apparently influenced by proximity, as the site closest to the 5'-splice site was greatly preferred. Studies with strains carrying some U1 snRNA mutations showed an increase in the use of the distal 3'-splice site, indicating a role for U1 snRNP in 3'-splice site selection. The data are compared with those from mammalian splice site choice experiments and suggest mechanisms that influence differential splice site choice as well as exon skipping.

To study the contribution of the large, 568-nucleotide yeast (Saccharomyces cerevisiae) U1 snRNA to pre-mRNA splicing, we generated mutations in two regions of the molecule and introduced each mutant gene back into yeast as the sole copy of the U1 snRNA gene. We mutagenized the "A loop," a subregion highly conserved in primary sequence in all U1 snRNA molecules analyzed to date. We also mutagenized a portion of the yeast core subdomain, a region conserved in primary and secondary structure among several yeast species but absent from the much smaller metazoan U1 molecule. Surprisingly, mutations in these two regions had little or no effect on growth rate, yet several of them affected an inefficiently spliced reporter gene construct. In addition, combinations of mutants in both regions gave rise to reduced growth rates. Using the latter assay, we confirmed some of the proposed secondary structure of the yeast core domain. The experiments indicate that both regions contribute to U1 snRNP activity but that mutations in a single region do not have a substantial effect on growth rate because U1 snRNP activity is not rate-limiting for growth.

The temporal and spatial expression of the period gene of Drosophila melanogaster has been analyzed by examining the expression of a per beta-galactosidase fusion gene in transformants and by in situ hybridization experiments with wild-type flies. Several strains of Drosophila melanogaster, transformed with the fusion gene, have been generated. The gene is active in mid-late embryos in the midline of the nervous system. Thereafter, beta-galactosidase activity is undetectable until the pupal stage when the prothoracic gland-corpora allata and the optic lobes are beta-galactosidase positive. In adults a surprisingly large number of tissues stain positively, including antennae, proboscis, eyes, optic lobes, cells of the central brain, cells of the thoracic ganglia, gut, Malpighian tubules, and ovarian follicle cells. The temporal pattern of expression agrees well with previous estimates made from developmental Northern blots with RNA extracted from wild-type animals. We suggest that many of the tissues that express the per gene contain their own intrinsic oscillator activity.

To investigate the importance of sequences between the yeast (Saccharomyces cerevisiae) branch point (TACTAAC box) and 3' splice site (AG), we generated a series of pre-mRNA substrates that differed in the length of RNA retained on the 3' side of the TACTAAC box. These pre-mRNAs were compared as substrates for the first step of in vitro splicing (5' cleavage and lariat formation) and in vitro spliceosome assembly (complex formation) in a whole-cell yeast extract. The results indicate that for rp51A pre-mRNA at least 29 nucleotides of RNA on the 3' side of the TACTAAC box are required for 5' cleavage and lariat formation, as smaller substrates fail to manifest any detectable cleavage or ligation events. Analysis of splicing complex assembly indicates that these smaller substrates undergo efficient yet incomplete complex formation; they are blocked at a late stage of spliceosome assembly, the complex I to complex II transition (Pikielny et al. 1986), a result which suggests that the failure to form lariats is due to a specific assembly defect. The lariat formation block (and assembly defect) can be relieved by the addition of ribohomopolymer "tails" to the 3' end of the shortened rp51A pre-mRNAs, and similar results were obtained with shortened actin pre-mRNAs. The results of this study indicate that this region of the pre-mRNA serves a specific function late in in vitro spliceosome assembly.

A chemical modification/interference assay was used to determine the yeast pre-mRNA sequence requirements for in vitro spliceosome assembly and splicing. Modifications of any of the nucleotides within the 5' splice site and branch point (TACTAAC box) consensus sequences as well as less conserved intron and exon positions were found to inhibit assembly and/or splicing. The interference pattern of the 5' splice site and TACTAAC box lesions increased as spliceosome assembly proceeded (complex III----complex I----complex II) and as splicing proceeded, suggesting that these sequence elements play multiple roles in the assembly of yeast spliceosomes and in the removal of intervening sequences. Furthermore, modification (or mutation) of a TACTAAC-like sequence upstream of the branch point was found to inhibit the rate of spliceosome assembly, implying a possible role for degenerate branch point sequences in modulating the efficiency of spliceosome assembly.