To determine whether the U1 snRNP accumulates cotranscriptionally and is detectable by ChIP, we constructed strain YKK19 (Table ), harboring tagged copies of endogenous Pol II (Myc-Rpo21p) and Prp42p (HA-Prp42p), a U1 snRNP-specific protein (34
). Figure shows schematically the proposed binding of the tagged U1 snRNP to nascent RNA, which is in turn tethered to the DNA axis by Pol II. Tagging of both essential proteins had no effect on the growth rate of the strain (data not shown), indicating that the normal functions of the Prp42p and Pol II were not disrupted. Two endogenous, intron-containing genes, ASC1
(Fig. ), were initially selected for analysis by ChIP because they are highly transcribed and have relatively large first exons (>500 bp) (19
). The DNA shearing procedure abolished PCR detection of ≥1-kb stretches along the genes of interest while preserving detection of ≤400-bp stretches (data not shown). Therefore, if the U1 snRNP associates with these genes, the system should resolve signals before and after intron synthesis.
FIG. 1. Detection of the U1 snRNP at intronic and downstream segments of DBP2 and ASC1 genes by ChIP. (A) Experimental design. In an endogenous transcription unit, the tagged U1 snRNP (Prp42p or Nam8p, triangle) is tethered to the chromatin by nascent RNA via (more ...)
Three gene regions from both DBP2
were well represented in the ChIPs of Pol II, whether the anti-Myc tag antibody (Fig. , lanes 10 to 12; Fig. , lanes 4 and 9) or an antibody against Pol II itself (Fig. , lanes 7 to 9; Fig. , lanes 5 and 10) was used. In agreement with previous results with tagged versions of various subunits of Pol II (24
), the distribution of Pol II along both genes was found to be fairly uniform, usually decreasing slightly downstream from the promoter. PCR products specifically resulted from ChIP of Pol II, since the same products were detected only very weakly in the nonimmune control ChIP (Fig. , lanes 2 and 3; Fig. , lanes 2 and 7), and Pol II was not detected at either the transcriptionally repressed GAL1
gene or the untranscribed telomere VI R (see Fig. and ). In contrast, HA-Prp42p ChIP templates yielded only very low levels of PCR product corresponding to either of the promoter regions, whereas intron and exon 2 regions of both genes were well detected (Fig. , lanes 4 to 6; Fig. , lanes 3 and 8). Quantitation of PCR products within the linear range (see Materials and Methods) revealed that Prp42p ChIPs contained 6.4 ± 1.1 (mean ± SEM, n
= 4 independent experiments)-fold-higher levels of DBP2
intron DNA than promoter-proximal DNA compared to Pol II ChIP templates prepared in parallel. Similarly, the DBP2
second exon was detected at 6.3 ± 2.4 (n
= 4)-fold-higher levels than the promoter region. We conclude that Prp42p is concentrated in downstream regions of both DBP2
genes both during and after intron synthesis.
FIG. 3. U1 snRNP levels remain constant along intronless genes. (A) ChIP analysis of three representative intronless genes—RPS3, ADH1, and PDR5—comparing promoter regions with downstream regions in the YKK19 strain. The data shown here are representative (more ...)
FIG. 4. Accumulation of the U1 snRNP on DBP2 depends on active transcription. Strain YKK20 contains a temperature-sensitive Pol II (rpb1-1) and an HA-tagged prp42p. When ChIP was performed on YKK20 grown at permissive temperature (24°C), the distribution (more ...)
To confirm that Prp42p detection reflects the cotranscriptional accumulation of the U1 snRNP, we performed ChIP experiments with strains harboring other tagged versions of U1 snRNP-specific proteins. First, we examined protein A-tagged Prp42p and Nam8p, a second U1-specific protein (16
). These strains were used previously to show that both PA-Prp42p and PA-Nam8p associate with the U1 snRNA by immunoprecipitation (16
) and to study the interaction of PA-Nam8p with pre-mRNA in commitment complex formation (43
). Figure shows that ChIP with rabbit IgG-coated beads preferentially selects the ASC1
intron region relative to the promoter in both strains. This experiment also indicates that the HA tag introduced into YKK19 does not produce different results from other tagged versions. Second, HA-tagged Prp40p, another U1 snRNP component which has been shown to bind hyperphosphorylated Pol II CTD by Far Western analysis (38
), was detected on DBP2
in an identical pattern to Prp42p (data not shown).
The present observation of U1 snRNP accumulation in downstream regions of ASC1
contrasts strongly with previous studies of the capping enzymes Ceg1p, Cet1p, and Abd1p, which have been detected at promoter regions of transcriptionally active genes (24
). Note that, unlike Abd1p, which remains associated with downstream regions, Ceg1p and Cet1p are preferentially concentrated in promoter regions (24
). To facilitate a direct comparison between U1 snRNP and capping enzyme dynamics on ASC1
, ChIP was performed by using a strain containing TAP-tagged Ceg1p, the mRNA guanylyltransferase (Fig. ). As expected, TAP-Ceg1p was highly concentrated at the promoter regions of the intronless gene PDR5
assayed in a previous study (24
). Similarly, TAP-Ceg1p was concentrated on both ASC1
promoter regions, verifying the differential distribution of capping and splicing factors on two intron-containing genes.
FIG. 2. Ceg1p is concentrated at the promoters of PDR5, ASC1, and DBP2. The distribution of the TAP-tagged mRNA guanylyltransferase, Ceg1p, was compared in promoter and downstream regions of three genes (see gene diagrams). Input lanes 1, 4, and 7 show the starting (more ...)
To address the possibility that U1 snRNP may accumulate in downstream regions of all genes, we assayed U1 snRNP and Pol II distributions along three intronless genes. ChIP with anti-Myc-Pol II or anti-Pol II showed robust signals for promoter and downstream regions in RPS3, ADH1, and PDR5 genes (Fig. , lanes 4, 5, 9, 10, 14, and 15). In contrast, HA-Prp42p was detected at very low levels relative to the control at every position along RPS3 and PDR5, while somewhat higher levels of HA-Prp-42p were observed on ADH1 (Fig. lanes 3, 8, and 13). Relative to levels obtained with Pol II ChIP templates, signals from HA-Prp42p ChIP either decreased or remained the same along the length of each gene. Therefore, U1 snRNP accumulation on DBP2 or ASC1 downstream regions cannot be attributed to generic changes in affinity for elongating Pol II, nonspecific binding to nascent RNA, or recruitment by the 5′ cap.
Because a low level of U1 snRNP association was detected within intronless genes relative to the nonimmune controls (Fig. , compare lanes 2 and 3, 7 and 8, and 12 and 13), we sought to determine whether gene induction is sufficient for U1 snRNP accumulation. GAL1 gene transcription was induced and changes in Prp42p association with the GAL1 promoter were determined. When YKK19 was grown in glucose, Pol II was detected at the ADH1 promoter but not on GAL1 or a transcriptionally inactive telomeric region on chromosome VI-R (Fig. , lane 4). Low levels of HA-Prp42p were detected at ADH1 only (Fig. , lane 3), as expected (see Fig. ). After 5 h of growth in galactose, Pol II was present on both ADH1 and GAL1, but Prp42p was not detected on the GAL1 promoter (Fig. , lanes 7 and 8) or in the downstream region (data not shown) relative to the nonimmune control. This suggests that U1 snRNP detection on chromatin is not due to transcriptional activity per se.
If U1 snRNP accumulation reflects specific binding to cognate sites in nascent RNA as proposed (Fig. ), then it is expected to depend on RNA synthesis. To test this prediction, we introduced an HA tag into the endogenous copy of Prp42p in strain DBY120, harboring the temperature-sensitive rpb1-1
allele of the Pol II large subunit (35
). A previous study showed that in this strain the Pol II holoenzyme dissociates from previously active transcription units when the cells are shifted to the nonpermissive temperature (46
). As expected, Pol II was detected at the ADH1
promoter and along DBP2
in DBY120 cells grown at 24°C but not at GAL1
or the telomere VI R (Fig. ). After 45 min of growth at 37°C, Pol II was not detectable above background levels at any gene region tested (Fig. , lanes 7 and 15). Similarly, the U1 snRNP was detectable on the intron and exon 2 regions of DBP2
promoter in DBY120 cells grown at 24°C but was undetectable after the shift to 37°C (Fig. , lanes 4, 8, 12, and 16). A similar loss of Pol II and U1 snRNP detection at 37°C was observed for ASC1
(data not shown). These data indicate that active transcription is required for U1 snRNP accumulation and that the U1 snRNP is not associated with chromatin independent of transcriptional activity.
To test whether association of the U1 snRNP with downstream regions depends on the intron, we assayed U1 snRNP levels along the DBP2
gene in a strain lacking the DBP2
intron. In this strain, the endogenous DBP2
ORF was replaced by the DBP2
cDNA; transcription levels of this intronless gene were previously found to be twofold higher than wild-type levels (2
). We had previously detected elevated U1 snRNP levels on both the intron and second exon of wild-type DBP2
(see Fig. ). Figure shows that removal of the intron abolishes accumulation of the U1 snRNP on downstream DNA corresponding to the second exon. Normalizing to Pol II ChIP signals, the ratio of Prp42p downstream to Prp42p at the promoter was only 0.68 (versus 6.3-fold in wild-type). Thus, despite Pol II-driven expression, elevated U1 snRNP levels were not observed at the DBP2
allele lacking its intron.
FIG. 5. Deletion of the DBP2 intron abolishes cotranscriptional U1 snRNP accumulation. Prp42p was tagged at the C terminus with an HA tag in strain yIB37 that contains the DBP2 cDNA in place of the endogenous intron-containing ORF (2). As expected, no intron (more ...)
Higher levels of U1 snRNP were detected on the DBP2
gene when the allele contained an intron (Fig. and ). Therefore, we postulated that intron-containing genes, constituting only ~5% of yeast ORFs, might accumulate more U1 snRNP than intronless genes. To test this directly, we performed genome localization analysis (20
). Cy3- and Cy5-labeled probes were generated from sheared YKK19 genomic DNA and HA-Prp42p and Myc-Pol II ChIP templates by linker-mediated PCR and hybridized with microarrays representing 6,229 yeast ORFs. The ratios of Cy3 and Cy5 median fluorescence intensities were used to analyze the data (see Materials and Methods). Microarrays hybridized with Cy3-genomic DNA versus Cy5-genomic DNA revealed a normal distribution of the data, as expected (Fig. , top panel). Interestingly, the data obtained from microarrays probed with HA-Prp42p ChIP templates versus genomic DNA yielded a major peak and a minor second peak of several hundred ORFs in each experiment, reflecting relatively higher scores for the labeled HA-Prp42p ChIP template probe (Fig. , center panel). Similarly, a second outlying peak was obtained from microarrays probed with Myc-Pol II ChIP templates and genomic DNA (Fig. , bottom panel). The striking difference between the shapes of the curves obtained with both ChIP templates compared to the genomic-genomic distribution indicates that specific sets of ORFs were selected by HA-Prp42p and Myc-Pol II ChIPs.
FIG. 6. Genome localization analysis of HA-Prp42 and Myc-Pol II. Cy3- and Cy5-labeled probes were synthesized from sheared genomic DNA or ChIP templates by linker-mediated PCR and hybridized with microarrays representing 6,229 yeast ORFs. Frequency histograms (more ...)
It was likely that the second outlying peaks obtained with HA-Prp42p and Myc-Pol II ChIP templates represented the population of ORFs associated with relatively high concentrations of Prp42p and Pol II. The mean values and the SD were determined for each experiment, and datum points >2 SD away from the mean were selected for further analysis. Results from five experiments comparing HA-Prp42p-genomic DNA and three experiments comparing Myc-Pol II-genomic DNA showed a high degree of reproducibility in the ORFs identified, by using the “2 SD criterion.” A list of HA-Prp42 ORF hits is provided in Table A1. Of the 388 ORFs hit in the HA-Prp42p arrays, 77 ORFs were hit every time (100%) and 161 ORFs were hit more than once (≥40%, Table ). Of 373 ORFs hit in the Myc-Pol II arrays, 234 occurred more than once (≥67%, Table ). A comparison of the Myc-Pol II hits with transcriptional frequency data (19
) revealed that the outlying peak indeed contained highly transcribed ORFs (data not shown).
ORFs selected by genome localization analysis
To determine whether the outlying peak observed with the HA-Prp42p ChIP templates was enriched in intron-containing ORFs, all ORFs were evaluated according to the yeast introndatabase
). Because genome localization analysis has the potential to detect ORFs on either the Watson or the Crick strand and because the resolution of the ChIP assay is ~400 bp, each ORF hit was also examined with respect to its position within the genome to determine whether introns occurred <500 nt away from the ORF, on either the same or opposite strand of DNA. If an ORF hit contained an intron or was found to be proximal to an intron-containing gene by the above criteria, the hit was scored as intron containing. Table shows the results obtained for all of the ORFs hit more than once in the HA-Prp42p and Myc-Pol II microarrays. For the ORFs hit in every HA-Prp42p experiment, 92% were intron containing compared to 51% for Myc-Pol II. In contrast, only 4.2% of the ORFs distributed outside 2 SD for the genomic-genomic distribution were intron containing, reflecting the fact that only 5% of the yeast ORFs contain introns. Thus, we conclude that the HA-Prp42p ChIP template is highly enriched for intron-containing ORFs with respect to the genome overall.
The yeast genome is predicted to contain 239 to 255 spliceosomal intron-containing ORFs (http://www.cse.ucsc.edu/research/compbio/yeast_introns.html
Al-though we detected 118 intron-containing genes by genome localization of Prp42p, we did not detect all of them. Because U1 snRNP accumulation is transcription dependent (Fig. ), some intron-containing genes may not be expressed at levels high enough to be detected in the outlying peak. However, because the array was produced by using oligonucleotide pairs to amplify ≤1 kb of the 3′ end of each ORF (see Materials and Methods), we also considered the possibility that genes containing relatively long second exons may exhibit diminished U1 snRNP accumulation in the 3′ regions represented on the arrays, either because the U1 snRNP has already left the nascent mRNP due to spliceosome assembly or because the tags become inaccessible to antibodies in downstream gene regions. To address this concern, we examined U1 snRNP accumulation on upstream and downstream regions of two such genes, ECM33
. Both of these genes contain introns very close to their promoters (Fig. ), and indeed significant U1 snRNP accumulation was detected in promoter-proximal regions by ChIP (Fig. , lanes 3 and 7). Interestingly, HA-Prp42p detection was reduced by ~70% in downstream regions of both genes. Neither ORF was well represented in the HA-Prp42p microarray results (Table ), suggesting that other ORFs with relatively long second exons might not have been detected by the microarray analysis performed here.
FIG. 7. Reduced detection of the U1 snRNP on downstream regions of intron-containing genes with long second exons. PCR detection of the 5′ and 3′ ends of ECM33 and SAC6 genes in the YKK19 strain comparing levels of Pol II and Prp42p. A schematic (more ...)
Genome localization data for genes examined in this study