Uridine-rich small nuclear RNAs (snRNAs) are the basal components of the spliceosome and play essential roles in splicing. The biogenesis of the majority of snRNAs involves 3′ end endonucleolytic cleavage of the nascent transcript from the elongating DNA-dependent RNA ploymerase II. However, the protein factors responsible for this process remain elusive in plants. Here, we show that DEFECTIVE in snRNA PROCESSING 1 (DSP1) is an essential protein for snRNA 3′ end maturation in Arabidopsis. A hypomorphic dsp1-1 mutation causes pleiotropic developmental defects, impairs the 3′ end processing of snRNAs, increases the levels of snRNA primary transcripts (pre-snRNAs), and alters the occupancy of Pol II at snRNA loci. In addition, DSP1 binds snRNA loci and interacts with Pol-II in a DNA/RNA-dependent manner. We further show that DSP1 forms a conserved complex, which contains at least four additional proteins, to catalyze snRNA 3′ end maturation in Arabidopsis. The catalytic component of this complex is likely the cleavage and polyadenylation specificity factor 73 kDa-I (CSPF73-I), which is the nuclease cleaving the pre-mRNA 3′ end. However, the DSP1 complex does not affect pre-mRNA 3′ end cleavage, suggesting that plants may use different CPSF73-I-containing complexes to process snRNAs and pre-mRNAs. This study identifies a complex responsible for the snRNA 3′ end maturation in plants and uncovers a previously unknown function of CPSF73 in snRNA maturation.
Uridine-rich small nuclear RNAs (snRNAs), ~60–200 nucleotide (nt) in length, are conserved noncoding RNAs in eukaryotes [1,2]. As the RNA components of the spliceosome, snRNAs (U1, U2, U4, U5, and U6) play essential roles in spliceosome formation and splicing of pre-messenger RNAs (pre-mRNAs) [1–3]. Most snRNAs are derived from their primary transcripts (pre-snRNAs) generated by DNA-dependent RNA polymerase II (Pol II), with the exception of Pol III-dependent U6 [4–7]. Like pre-mRNAs, pre-snRNAs are transcribed beyond the 3′ end of mature snRNAs [4,8, 9]. Consequently, pre-snRNAs subject to 3′ maturation, a process involving endonucleolytic cleavage of the nascent transcript from the elongating polymerase in the nucleus followed by a 3′-to-5′ exonucleolytic trimming step in the cytoplasm [4,8, 9]. Previous studies have identified three elements required for proper 3′ end cleavage of Pol II-dependent snRNAs in metazoans: an snRNA promoter containing the distal sequence element (DSE) and the proximal sequence element (PSE), the C-terminal domain (CTD) of Rpb1 (the largest subunit of Pol II), and the 3′ box that localizes the downstream of the cleavage site [5,8–13]. In metazoans, the integrator complex (INT), which contains at least 14 subunits, is responsible for pre-snRNA 3′ end cleavage [14]. Among INT subunits, INT1,4, 9, and 11 are essential for snRNA 3′ processing, whereas INT3 and 10 are dispensable for maturation [14,15]. INT11 is a paralog of the cleavage and polyadenylation specificity factor 73 kDa (CPSF73), which is the catalytic component of the CPSF complex that cleaves mRNAs, but not snRNAs, at the 3′ end [14]. Because of this, INT11 was proposed to cleave pre-snRNA at 3′ end [14,16]. INT requires Pol II and the promoter elements for its recruitment to snRNA loci [6,17–21]. However, it is not clear how INT specifically recognizes snRNA loci and transcripts. Yeast uses different mechanisms to process the snRNA 3′ end because it does not contain INT, and its snRNA gene structures differ from their metazoan counterparts [6,7]. In plants, the major Pol II-dependent snRNAs include U1, U2, U4, and U5 [22–29]. Each of them has more than ten copies in the Arabidopsis genome [30]. Although plant snRNA promoters have diverged from their metazoan counterparts and do not contain DSE and PSE, they do have an upstream sequence element (USE) and a proximal TATA box, which are conserved and essential for their transcription [25]. Plant snRNA genes have a conserved 3′ box (CA (N) 3-10AGTNNAA) downstream of mature snRNAs, which is necessary for snRNA processing [27,31]. In plants, the processing of snRNAs can be uncoupled from transcription initiation, because their promoters are not required for 3′ end cleavage [31]. In addition, many subunits of INT, including INT11 and the putative scaffold protein INT1, are missing in plants [16], suggesting that plants may use a mechanism different from that of metazoans to process snRNA 3′ end. Here, we report that snRNA 3′ end maturation in Arabidopsis requires a protein named DEFECTIVE in snRNA PROCESSING 1 (DSP1). DSP1 binds snRNA loci and interacts with Pol II in a DNA/RNA-dependent manner. A hypomorphic dsp1-1 mutation causes pleiotropic developmental defects, impairs snRNA 3′ maturation, and alters the occupancy of Pol II at snRNA loci. DSP1 forms a conserved complex with DSP2, DSP3, DSP4, and CPSF73-I to process snRNAs. Unlike CPSF73-I, which is also the catalytic component of the plant CPSF complex, the DSP1 complex does not affect mRNA 3′ maturation. Based on these results, we propose that two CPSF73-I complexes separately process pre-snRNAs and pre-mRNAs in Arabidopsis. This study identifies an snRNA-processing complex and uncovers an unknown function for CPSF73 in plants.
In order to identify proteins involved in snRNA maturation in Arabidopsis, we screened for mutants containing increased levels of pre-U2. 3 snRNA (At3g57765) from a T-DNA collection obtained from the Arabidopsis Stock Center. We reasoned that impaired snRNA 3′ end cleavage should increase the levels of pre-snRNAs. From ~ 500 T-DNA insertion lines, we identified a mutant (Salk_036641C) containing elevated levels of pre-U2. 3 snRNA relative to wild-type plants (WT; Columbia-0 [Col]) through reverse transcription PCR (RT-PCR) analyses (Fig 1A and S1 Data). We named this mutant defective in snRNA processing 1–1 (dsp1-1). In dsp1-1, a T-DNA insertion in the second intron of At4g20060 (DSP1) reduced the transcript levels of DSP1 (S1A–S1C Fig). However, dsp1-1 showed incomplete penetrance, as only a portion of plants showed increased levels of pre-U2. 3 snRNA, accompanied with pleiotropic development defects such as smaller size, delayed flowering, reduced fertility, and enlarged cell size (Fig 1B and S1D–S1F Fig). To demonstrate that dsp1-1 is responsible for the observed phenotypes, we crossed dsp1-1 to DSP1/dsp1-2 (CS16199), which contains a T-DNA insertion in the sixth exon of DSP1 (S1A and S1B Fig). The F1 dsp1-1/dsp1-2 mutant displayed more severe growth defects and higher levels of pre-U2. 3 snRNA than dsp1-1 (Fig 1C and S1G Fig, S1 Data). Furthermore, a WT copy of DSP1 driven by its native promoter (pDSP1: : DSP1-Green Fluorescent Protein [GFP]) in dsp1-1 rescued the developmental defects and restored the levels of pre-U2. 3 snRNA (Fig 1A and 1B, S1 Data), demonstrating that DSP1 is required for plant development and may be involved in snRNA biogenesis. We suspected that the dsp1-2 mutation might cause embryo lethality, because the homozygous dsp1-2 mutant could not be obtained, and aborted seeds were observed in siliques of DSP1/dsp1-2 (Fig 1D). In fact, Nomarski microscopy showed embryos, whose terminal phenotype arrested at the globular stage, in the siliques of DSP1/dsp1-2 (S1H Fig). Agreeing with this result, most dsp1-1/dsp1-1 seeds displayed delayed embryo development relative to WT (Fig 1E). Furthermore, a small portion of dsp1-1 seeds contained abnormal embryos (S1I Fig), suggesting that dsp1-1 might impair cell division and/or pattern formation. We also found that the transmission of dsp1-2 was reduced, as the ratio of DSP1/dsp1-2 versus WT (1: 1. 3) was less than the expected ratio (1: 1) in offspring of DSP1/dsp1-2. To determine whether DSP1 influences male or female gametophyte transmission, we performed reciprocal crosses between DSP1/dsp1-2 and WT and analyzed transmission of dsp1-2. When WT was used as a pollen donor, dsp1-2 was transmitted normally (S1 Table). However, when DSP1/dsp1-2 was used as a pollen donor, the transmission rate of dsp1-2 was reduced (S1 Table), suggesting that dsp1-2 might affect male gametophyte transmission. In order to examine how dsp1 influences male gametophyte transmission, we first examined pollen viability using Alexander' s staining. Although pollens from WT appeared full, round, and red-stained, many pollens from dsp1-1 could not be stained (Fig 1F), suggesting that they are completely or partially devoid of cytoplasmic content, indicative of a defect in pollen viability. We also examined pollen germination and tube growth of the viable dsp1-1 pollen grains but did not observe obvious differences from WT (S1J and S1K Fig). These results suggest that DSP1 participates in male gametophyte transmission by influencing pollen viability. Because the structures of Pol II-dependent snRNA genes share considerable similarities [30], we hypothesized that DSP1 might have a general effect on pre-snRNA levels. To test this hypothesis, we randomly selected several Pol II-dependent pre-snRNAs from the U1, U4, and U5 gene families and examined their abundance in WT and dsp1-1 by qRT-PCR and RT-PCR. The accumulation of these selected pre-snRNAs (pre-U1a, pre-U2. 3, pre-U4. 2, and pre-U5. 6 snRNAs) was much higher in dsp1-1 than that in WT, which was rescued by the GFP-DSP1 transgene (Fig 2A and S2A Fig, S1 Data). In contrast, the abundance of Pol III-dependent pre-U6. 26 snRNA was not affected by dsp1-1 (Fig 2A and S2A Fig, S1 Data). These results suggest that DSP1 likely has a general role in the biogenesis of Pol II-dependent snRNAs. We further examined the effect of dsp1-1 on the accumulation of mature U1 and U2 snRNAs using northern blot. As observed in metazoans [14], the abundance of mature U1 and U2 RNAs in dsp1-1 was comparable to that in WT (Fig 2B), which could be explained by the facts that dsp1-1 is a hypomorphic mutation and snRNAs have a long half-life [32]. Cloning and sequencing analyses further showed that mature U2 RNAs were proper processing products (S2B Fig). RNase protection assay showed the increased accumulation of pre-U1 and pre-U2 snRNAs in dsp1-1 and confirmed the results obtained from northern blot (S2C and S2D Fig). Consistent with its effect on mature snRNAs, dsp1-1 did not impact the splicing of several examined mRNAs (S2E Fig). The increased pre-snRNA levels in dsp1-1 could result from defection in pre-snRNA 3′ end cleavage or increased pre-snRNA transcription. To distinguish these two possibilities, we first evaluated if dsp1-1 influenced pre-U2. 3 snRNA 3′ end cleavage with an in vitro assay using the U2. 3 gene as reporter according to [13]. In this assay, a 5′ end [P32]-labeled pre-U2. 3 snRNA was processed in nuclear proteins extracted from inflorescences of dsp1-1 or WT. We also included a pre-U2. 3 snRNA with a poly-G tail at 3′ end (pre-U2. 3-pG), which prevents 3′ trimming activity [33], to rule out the possibility that the product is generated from the 3′ end trimming rather than endonucleolytic cleavage. The accumulation of U2. 3 snRNAs (~196 nt) generated from both pre-U2. 3 and pre-U2. 3-pG was reduced in dsp1-1 relative to their levels in WT at various time points (Fig 2C and S2F Fig). Quantification analysis of the 90-min reaction showed that the overall pre-U2. 3 snRNA processing activity in dsp1-1 was approximately 40% of that in WT (Fig 2D and S1 Data). In addition, the DSP1-GFP transgene restored pre-U2. 3 snRNA processing in dsp1-1 (Fig 2C and 2D, S1 Data). These results suggest that DSP1 might be required for the snRNA 3′ end maturation. To further test the effect of DSP1 on snRNA transcription and 3′ maturation, we used an in vivo GUS reporter gene assay. In this assay, the GUS gene was fused to the 3′ end of the U2. 3 gene that contains the promoter, the coding region, and 3′ box region (pU2: : pre-U2-GUS; Fig 2E) according to [15]. If properly cleaved, pre-U2-GUS RNAs would not be translated into GUS protein (Fig 2E), whereas disrupted cleavage would result in GUS accumulation. As a control for transcription, we generated a GUS reporter fused with a mutated U2. 3 gene (pU2: : pre-U2m-GUS), in which the 3′ box was mutated to disrupt pre-U2 snRNA processing (Fig 2F), with expectation that the GUS protein would be accumulated (Fig 2E and S2G and S2H Fig). The alteration of pre-U2m-GUS levels in dsp1-1 relative to WT would reflect the effect of DSP1 on pre-snRNA other than cleavage. Transgenic lines expressing pU2: : pre-U2-GUS or pU2: : pre-U2m-GUS were generated in a Col background and subsequently crossed to dsp1-1 (S2H Fig). In F2, DSP1+ (DSP1/DSP1 or DSP1/dsp1-1), or dsp1-1, genotypes containing the GUS transgene were identified through PCR genotyping. GUS activities and the abundance of pre-U2m-GUS transcripts were slightly reduced in dsp1-1 relative to DSP1+ (Fig 2F and 2G and S2I Fig, S1 Data; bottom panel), suggesting that dsp1-1 does not increase the transcription of pre-snRNAs. In contrast, relative to DSP1+, the GUS activities and pre-U2-GUS transcript levels were increased in various tissues of dsp1-1 harboring pre-U2-GUS (Fig 2F and 2G and S2I Fig, S1 Data; top panel). These results demonstrate that DSP1 is essential for snRNA 3′ end cleavage. In metazoans, the INT complex co-transcriptionally processes pre-snRNAs [16]. This led us to hypothesize that DSP1, if it has a direct role in snRNA processing, might be a nuclear-localized protein that associates with the snRNA loci. To examine the subcellular localization of DSP1, we expressed GFP-DSP1 from the CaMV35S promoter (35S: : GFP-DSP1) in leaf epidermal cells of Nicotiana benthamiana. In these cells, GFP-DSP1 localized to the nucleus (Fig 3A). Consistent with this result, GFP-DSP1 was detected in the nuclear protein fraction, but not in the cytoplasmic protein fraction (Fig 3B), both of which were extracted from the dsp1-1 harboring 35S: : GFP-DSP1. To examine the association of DSP1 with the U2. 3 locus, we performed a chromatin immunoprecipitation (ChIP) assay using dsp1-1 harboring GFP-DSP1 or GFP (negative control) and then checked the presence of the U2. 3 locus in the ChIPs of GFP-DSP1 and GFP IPs using PCR and quantitative PCR (qPCR). The USE, TATA box (U2-TA), coding region (U2-C), and 3′ box (U2-3′ box; the highest signal) of the U2. 3 locus were enriched in the ChIPs of GFP-DSP1, but not in the ChIPs for GFP, relative to “no-antibody” controls (Fig 3C and 3D and S3A Fig, S1 Data). In addition, the downstream regions (U2-DS1 and U2-DS2) of the 3′ box of the U2. 3 locus and the ACTIN2 locus (Pol II-dependent) were not enriched in the ChIPs of GFP-DSP1 (Fig 3C–3E and S3A and S3B Fig, S1 Data). DSP1 also occupied the USE, TATA-box, coding region, and 3′ box (the highest signal) of the U1a locus, but not in the downstream regions (U1-DS1 and U1-DS2) of the U1a 3′ box (Fig 3F and S3C Fig, S1 Data). These results show the occupancy of DSP1 at the snRNA loci, which, together with the fact that dsp1-1 causes the defection of snRNA processing, demonstrates that DSP1 has a direct role in snRNA biogenesis. Both U1a and U2. 3 were transcribed through the DS1 region (S3D Fig). The absence of DSP1 in the DS1 region suggests that DSP1 may not travel through the 3′ box or be released at the 3′ box after cleavage. The occupancy of DSP1 at the snRNA loci prompted us to test the interaction between DSP1 and Pol II by a co-immunoprecipitation (Co-IP) assay [34]. GFP-DSP1 and RPB2 (the second-largest subunit of Pol II) were able to reciprocally co-IP (Fig 3G and 3H). In contrast, GFP did not interact with RPB2 (Fig 3G and 3H). In addition, GFP-DSP1 and RPB2 proteins were not detected in the “no-antibody” reactions. These results confirm a DSP1-Pol II association. We further examined the dependence of the DSP1–Pol II interaction on DNAs/RNAs. Treatments with either DNase I or RNase A reduced the interaction of DSP1 with Pol II (Fig 3I), whereas micrococcal nuclease, which acts on both RNAs and DNAs, abolished the DSP1–Pol II interaction (Fig 3J). We next evaluated the effect of dsp1-1 on Pol II occupancy at the U2. 3 locus in a ChIP assay using anti-RPB2 antibodies. As expected, Pol II occupied the U2. 3 and ACTIN2 loci, but not the Pol II C1 locus (an intergenic DNA fragment between At2g17470 and At2g17460) (Fig 3K and 3L) [35]. dsp1-1 reduced Pol II occupancy at the USE, TATA box, and U2. 3C of the U2. 3 locus, but not at the ACTIN2 locus (Fig 3K and 3L, S1 Data). Interestingly, dsp1-1 did not alter the occupancy of Pol II at the 3′ box (Fig 3K). dsp1-1 had a similar effect of Pol II occupancy at various regions of the U1a locus (Fig 3M and S1 Data). These results suggest that DSP1 is required for proper occupancy of Pol II at snRNA loci. DSP1 does not contain any known nuclease domains, suggesting that it may associate with other proteins to act in snRNA maturation. DSP1 is a conserved protein in higher plants and contains an N-terminal armadillo (ARM) -like fold (Fig 4A and S4A Fig), which arranges in a regular right-handed super helix that provides a solvent-accessible surface for binding large substrates, such as proteins and nucleic acids, and a C-terminal region of unknown function [36]. We found that the ARM domain of DSP1 shared ~25% similarity with that of the integrator subunit 7 (INT7) of metazoans (Fig 4A). This led us to suspect that an INT-like complex might exist in plants. If so, an INT11 (the catalytic subunit of INT) -like nuclease should function in snRNA processing in plants. Arabidopsis encodes two INT11-like nucleases, CPSF73-I and CPSF73-II, which are conserved in higher plants (S4B Fig) [37,38]. Because they lack the characteristic C-terminal region of INT11 (Fig 4A), which is essential for snRNA maturation [39], and act as the catalytic components of the CPSF complex to cleave pre-mRNA 3′ end [37,38], CPSF73-I and CPSF73-II were never thought to act on snRNA processing. However, CPSF73-I is essential for both pollen and embryo development [38]. This resembles the effect of DSP1 on plant development, suggesting that CPSF73-I might be the nuclease that processes snRNAs in plants. To test this, we used an artificial miRNA (amiRCPSF73-I) to knockdown the expression of CPSF73-I (Fig 4B and S4C Fig, S1 Data) [40]. The reduced expression of CPSF73-I in the amiRCPSF73-I lines caused developmental defects and increased the levels of pre-U2. 3 snRNAs (Fig 4C and 4D, S1 Data). Expression of an amiRCPSF73-I-resistant CPSF73-I (CPSF73-I-R) in the amiRCPSF73-I lines recovered the levels of pre-U2. 3 snRNA (Fig 4E and S1 Data), suggesting that CPSF73-I is required for snRNA 3′ end maturation. Next, we tested if CPSF73-II also had a role in pre-snRNA processing (S4C Fig). Although amiRCPSF73-II reduced the expression of CPSF73-II, resulting in pleiotropic developmental defects (S4C–S4E Fig and S1 Data), it did not affect the levels of pre-snRNAs (S4F Fig and S1 Data). We also examined whether CPSF100, which partners with CPSF73-I to process pre-mRNAs, is required for snRNA processing. However, a knockdown of CPSF100 by amiRCPSF100 did not alter the levels of pre-U2. 3 RNAs (S4C, S4G and S4H Fig and S1 Data). The above results suggest the presence of a CPSF73-I containing complex that acts on pre-snRNAs. Indeed, size-exclusion high performance liquid chromatography (HPLC) detected a ~670 kDa (eluted at 93–102 min) CPSF73-I-containing complex and a larger complex (eluted at 72–78 min) besides CPSF73-I monomers in the protein extracts of the transgenic plants harboring a 35S: : GFP-CPSF73-I transgene (S4I Fig). The ~670 kDa complex, but not the larger one, was able to process pre-U. 2 snRNAs (S4J Fig). Next, we tested if this ~670 kDa complex could act on pre-mRNAs using a 5′ end [P32]-labeled RNA (RSB-3; ~380 nt) that covers the 3′-UTR of an rubisco small subunit gene (At5g38420) [41]. Proper 3′ end processing of RSB-3 would generate a ~240 nt RNA fragment and a ~190 nt RNA fragment due to the presence of two poly (A) sites (S4L Fig) [41]. The 670 kDa complex did not process RSB-3 (S4L Fig). We also examined the effect of dsp1-1 on the formation of the CPSF73-I complex and its activity. The size of the snRNA-processing complex became smaller in dsp1-1 relative to that in Col, and its pre-U2. 3 snRNA processing activity was reduced (S4I and S4K Fig). In contrast, the larger complex was still intact in dsp1-1 (S4D Fig). We further examined the occupancy of CPSF73-I at the U2. 3 locus in N. benthamiana leaves harboring both GFP-CPSF73-I and pU2: : pre-U2-GUS. ChIP assay detected the occupancy of CPSF73-I at the pU2: : U2. 3-GUS gene, with the highest occupancy at the 3′ box (Fig 4F and S1 Data). We also tested the interaction of CPSF73-I with Pol II in Col harboring a 35S: : GFP-CPSF73-I transgene. However, unlike DSP1, CPSF73-I did not interact with Pol II (Fig 4G). Next we sought to identify additional proteins acting in snRNA maturation by searching for Arabidopsis homologs of other INT subunits. We identified At4g14590 (named DSP2), At3g08800 (named DSP3; also known as SHORT-ROOT INTERACTING EMBRYONIC LETHAL, SIEL) [42], and At3g07530 (named DSP4) as potential homologs of INT3, INT4, and INT9, respectively (Fig 5A). Among them, DSP2 is approximately half size of INT3 and shares ~57% similarity with the N-terminal fragment (aa, 1–490) of INT3 (Fig 5A). The ARM domain, but not other regions, of DSP3 shared similarities with INT4 (Fig 5A). DSP4 has ~46% similarity with INT9 (Fig 5A). Like DSP1 and CPSF73-I, these proteins are conserved in higher plants (S5A–S5C Fig). We evaluated if DSP2, DSP3, and DSP4 were required for snRNA processing using their loss-of-function mutants. The DNA knockout mutants for DSP2 (CS848944) and DSP3 (SALK_089544; dsp3-2; also known as seil-2) displayed embryo lethality (Fig 5B and S5D Fig), whereas expression of DSP4 was not altered in the available T-DNA insertion mutants (SALK_005904; S5D–S5F Fig). We thus obtained a weak allele of DSP3 (SALK_086160, dsp3-1; siel-4), in which a T-DNA insertion reduced the expression levels of DSP3 and constructed knockdown lines of DSP2 (amiRDSP2) and DSP4 (amiRDSP4) with artificial miRNAs (S5D–S5G Fig). dsp3-1, amiRDSP2, and amiRDSP4 reduced the expression of DSP3, DSP2, and DSP4, respectively, and caused pleiotropic developmental defects (Fig 5C–5E and S5F and S5H–S5I Fig, S1 Data). qRT-PCR showed that the levels of pre-U2. 3 snRNAs were increased in dsp3-1 and amiRDSP4 relative to those in WT (Fig 5F and 5G, S1 Data), suggesting that they might act in snRNA processing. However, the levels of pre-U2. 3 snRNAs were not altered or slightly lower in amiRDSP2 relative to those in WT (S5J Fig and S1 Data), agreeing with a dispensable role of INT3 for pre-snRNA maturation [15]. To confirm the role of DSP2, DSP3, DSP4, and CPSF73-I in snRNA maturation, we examined their effect on the 3′ end cleavage of pre-U2. 3 snRNAs using the in vitro processing assay. The accumulation of mature snRNAs generated from pre-U2. 3 and pre-U2. 3-pG was lower in nuclear protein extracts from dsp3-1, amiRDSP4, or amiRCPSF73-I than from WT (Fig 5H and 5I and S6A Fig, S1 Data). In contrast, amiRDSP2 did not impair pre-U2. 3 and pre-U2. 3-pG processing (Fig 5H and 5I and S6A Fig, S1 Data). In addition, the expression of CPSF73-I-R in the amiRCPSF73-I line fully recovered the processing of pre-U2. 3-pG snRNA (S6B Fig). These results demonstrate that, like DSP1 and CSPF73-I, DSP3 and DSP4 are required for snRNA processing. The involvement of CPSF73-I in both pre-mRNA and pre-snRNA processing raised the possibility that the DSP proteins might also function in pre-mRNA 3′ end cleavage. Therefore, we tested their effect on the 3′ end processing of the RSB-3 RNA (S4L Fig) using nuclear protein extracts. As expected, the 3′ end processing efficiency of RSB-3 was reduced in amiRCPSF73-I and amiRCPSF73-II relative to WT at various time points (Fig 5J–5L and S6C Fig, S1 Data). In contrast, RSB-3 processing in protein extracts from dsp1-1, dsp3-1, amiRDSP2, and amiRDSP4 was comparable with that of WT (Fig 5J–5L and S1 Data), suggesting that DSP1, DSP2, DSP3, and DSP4 are not required for pre-mRNA 3′ end processing. To further validate the result, we monitored the 3′ end formation of FCA mRNA, which is known to be affected by the pre-mRNA 3′ end processing complex [43], in dsp, amiRCPSF73-I, and amiRCPSF73-II by northern blot. amiRCPSF73-I and amiRCPSF73-II, but not dsp1-1, dsp3-1, amiRDSP2, and amiRDSP4, altered the 3′ end formation of FCA (S6D Fig). The involvement of DSP1, DSP3, DSP4, and CPSF73-I in the snRNA maturation raised the possibility that they may form a complex to cleave pre-snRNAs. To test this possibility, we first examined the interaction of DSP1 with the other proteins using co-IP. DSP2 was included in this experiment because its homolog INT3 is a component of the INT complex [14]. We also included CPSF100 as a control because it is a homolog of DSP4 but does not affect snRNA processing. GFP-DSP1 was transiently co-expressed with MYC-DSP2, MYC-CPSF100, MYC-DSP4, or MYC-CPSF73-I in N. benthamiana as described previously [34]. MYC-DSP4 and MYC-CPSF73I, but not MYC-DSP2 and MYC-CPSF100, were detected in the GPF-DSP1 precipitates (Fig 6A–6C and S7A Fig). In addition, the control, GFP, did not co-IP with MYC-DSP2, MYC-DSP4, and MYC-CPSF73-I (Fig 6A–6C). We were unable to express the recombinant DSP3 protein in either N. benthamiana or Escherichia coli, likely because it is extremely unstable. To test the interaction of DSP1 with DSP3, we generated a recombinant DSP3-MYC protein using an in vitro translation system as described [44]. However, DSP1 did not co-IP with DSP3-MYC (Fig 6D). These results support the interaction of DSP1 with DSP4 and CPSF73-I, but not with DSP2, DSP3, and CPSF100. We further tested the interaction of GFP-DSP2 with DSP3-MYC, MYC-DSP4, or MYC-CPSF73-I. GFP-DSP2 interacted with MYC-DSP4 but not with DSP3-MYC and MYC-CPSF73-I (Fig 6E–6G). Co-IP/pull down assays also showed that DSP4 did not interact with DSP3 and CPSF73-I, but DSP3 did interact with CPSF73-I (Fig 6H–6J). To confirm these protein interactions, we performed a bimolecular fluorescence complementation (BiFC) assay (S1 Text) [45]. In this assay, the paired proteins, which were fused to the N-terminal fragment of yellow fluorescent protein (nYFP) or to the C-terminal fragment of YFP (cYFP), respectively, were introduced into tobacco cells by infiltration. The interaction of the two protein partners will result in a functional YFP [34]. As expected, the DSP1–DSP4, DSP1–CPSF73-I, DSP2–DSP4 interactions, but not the DSP1–DSP2, DSP2–CPSF73-I, and DSP4–CPSF73-I interactions, were confirmed (S7B Fig). We further validated the protein interactions using stable transgenic lines harboring GFP-DSP1/MYC-CPSF73-I, GFP-DSP1/MYC-DSP4, or GFP-DSP4/MYC-CPSF73-I transgenes. As observed in tobacco, we detected the DSP1–DSP4 and DSP1–CPSF73-I interactions, but not the DSP4–CPSF73-I interaction, in Arabidopsis (Fig 6K–6M). Next, we asked if these proteins could co-exist in a complex. We found that GFP-DSP1 pulled down both CPSF73-I and DSP3 from protein extracts containing DSP3-MYC, GFP-DSP1, and HA-CPSF73-I (Fig 6N). In addition, when co-expressed, DSP4 co-IPed with DSP1, DSP2, and CPSF73-I, while CPSF73-I co-IPed with DSP1, DSP2, and DSP4 (Fig 6O and 6P). These results demonstrate that DSP1, DSP2, DSP3, DSP4, and CPSF73-I likely form a complex to process snRNAs (Fig 7).
We identified a conserved complex essential for 3′ end maturation of Pol II-dependent snRNAs in plants. This complex contains at least five proteins, including DSP1, DSP2, DSP3, DSP4, and CPSF73-I. In this complex, DSP1 bridges DSP4 and CPSF73-I, whereas DSP2 and DSP3 may act as accessory components of DSP4 and CPSF73-I, respectively (Fig 7). More importantly, we show that CPSF73-I likely is the catalytic component for snRNA 3′ end processing. This result shows that higher plants use the same enzyme to process both pre-mRNAs and pre-snRNAs. However, the two CPSF73-containing complexes might function separately in snRNA and pre-mRNA maturation (Fig 7), as the dsp mutations do not impair the mRNA 3′ end processing and a knockdown of CPSF-100 or CPSF73-II does not affect snRNA 3′ end maturation. Furthermore, mass spectrometry analyses did not identify any DSP proteins in the CPSF-100 complex [46]. Consistent with this, DSP1 interacts with DSP4 but not its homolog CPSF-100. In contrast to what we have discovered in plants, in metazoans, CPSF73 and its paralog, INT11, are used to process pre-mRNAs and pre-snRNAs, respectively. However, the similarities of some DSP proteins with their counterparts in INT raise the possibilities that a common ancestor complex containing CPSF73 might have been used to process pre-snRNAs before divergence between metazoans and plants and that CPSF73 may be subject to sub functionalization in metazoans. How does the DSP1 complex recognize and process pre-snRNAs? The occupancy of DSP1 and CPSF73-I at snRNA loci and the DSP1-Pol II association support the idea that the DSP1 complex processes pre-snRNAs co-transcriptionally. Both CPSF73 and DSP1 have the highest occupancy at the 3′ box, and mutations in the 3′ box greatly reduced the activity of the DSP1 complex, demonstrating that the 3′ box is essential for the DSP1 complex to recognize the cleavage site. In metazoans, Pol II plays key roles in recruiting INT to snRNA loci and transcription initiation is essential for snRNA processing [6,17–21]. However, in plants, blocking transcription initiation only has a minor effect on snRNA processing [31]. In addition, DSP1 interacts with Pol II in a DNA/RNA-dependent manner, whereas CPSF73-I and DSP4 do not associate with Pol II (Fig 4G and S5K Fig). These results suggest that Pol II is not crucial for recruiting the DSP1-CPSF73 complex to the snRNA loci, although we cannot completely rule out this possibility. Perhaps the DSP1 complex can recognize specific sequence in the promoters of snRNAs. Alternatively, the DSP1 complex might be recruited to snRNA loci through its interaction with some snRNA-specific transcription factors. Clearly, all these possibilities need to be examined in the near future. The DSP1 complex may have other roles in snRNA biogenesis. The facts that the DSP1 interacts with the snRNA promoters and that the dsp1-1 mutation reduced the occupancy of Pol II at the promoters and coding regions of U1 and U2 snRNA genes support that the DSP complex promotes the transcription of Pol-II dependent snRNAs. In further support of this, the transcript levels of preU2m-GUS RNAs are slightly lower in dsp1-1 than in WT (Fig 2H). However, it is not clear whether the DSP1 complex directly or indirectly regulates snRNA transcription. The DSP complex may also positively contribute to Pol II releasing at the snRNA 3′ end, because the 3′ end cleavage will help transcription termination. If so, the Pol II occupancy at the 3′ end of snRNA loci should be increased in dsp1-1. However, we observed unchanged Pol II occupancy at the 3′ end in dsp1-1 relative to Col. This result likely reflects the combined effects of DSP1 on snRNA transcription and 3′ end processing. Besides snRNA biogenesis, the DSP complex may have other functions, given the facts that lack of DSP2, which has a minor role in snRNA processing, causes embryo lethality and developmental defects (Fig 5) and that dsp1-1, in which the abundance of mature snRNAs is comparable to that of WT, still displays pleiotropic developmental defects (Figs 1 and 2). In fact, DSP3 (known as SIEL) has been shown to promote root patterning through interacting with SHR, a transcription factor, and promoting its movement [42]. It will be interesting to test whether other DSP components have similar functions in root patterning. In metazoans, INT not only functions in snRNA processing, but also controls the transcription termination of some mRNAs, the biogenesis of enhancer RNAs, which are noncoding RNAs regulating gene expression, and the biogenesis of some viral-derived miRNAs [33,47–50]. It is possible that the DSP complex plays similar roles in plants. We identified several mRNAs containing the 3′ box at their 3′ end from the Arabidopsis genome. However, the DSP complex does not affect their processing. Thus, it remains to be determined if the DSP complex has other substrates and, if so, what these substrates are.
T-DNA insertion mutants including CS848944, SALK_089544, SALK_005904, SALK_036641, CS16199, and SALK_086160 were obtained from the Arabidopsis stock center (www. arabidopsis. org); all are in the Col genetic background. Transgenic lines (Col background) harboring pU2: : pre-U2-GUS or pU2: : pre-U2m-GUS were crossed to dsp1-1. In the F2 population, DSP1+ (DSP1/DSP1; DSP1/dsp1-1) plants and dsp1-1 containing the transgenes were identified by genotyping of T-DNA and GUS using primers listed in S2 Table. A 6. 4 kb genomic fragment containing the DSP1 promoter and coding regions was PCR amplified, cloned into pENTR/SD/D-TOPO, and subsequently cloned into the binary vector pGWB4. The resulting plasmid was transformed into dsp1-1, and transgenic plants were screened for Hygromycin resistance. DSP1 cDNA was amplified by RT-PCR, cloned into pENTR/SD/D-TOPO, and subsequently cloned into pEG104 [51] to generate the 35S: : GFP-DSP1 fusion vector. A genomic fragment containing the U2. 3 gene promoter, snRNA coding region, and 3′ box region was PCR amplified and cloned into pMDC164 to generate pU2: : pre-U2-GUS. The 3′ box of pre-U2-GUS was then mutated to generate pU2: : pre-U2m-GUS using a Site-Directed Mutagenesis Kit (Stratagene). The primers used for plasmid construction are listed in S2 Table. Siliques of different developmental stages were dissected with hypodermic needles, mounted on microscope slides in a clearing agent (Visikol) overnight, and then observed with a confocal microscope. To visualize GUS expression, samples were immersed in the GUS staining solution for 12 h in the dark. The stained samples were treated with 70% ethanol to remove chlorophyll before observation using a dissecting microscope. cDNA was synthesized from 2 μg of total RNA with reverse transcriptase (Invitrogen) and random primers. qPCR was performed in triplicate on a Bio-Rad IQcycler apparatus with the Quantitech SYBR green kit (Bio-Rad). The primers used for PCR are listed in S2 Table. In vitro processing assays of pre-U2. 3 and the 3′ UTR of a Rubisco small subunit gene (RSB-3) were performed as described [13,41]. Briefly, DNA templates used for in vitro transcription of pre-U2. 3 and RBS-3 were amplified using T7 promoter-anchored primers (S2 Table). A 5′ end [32P]-labeled pre-U2. 3 snRNA was incubated with 2 μg nuclear proteins in a 20 μl reaction, while [32P]-labeled RSB-3 was cleaved by 4 μg nuclear proteins in a 20 μl reaction. After reactions were stopped at various time points, RNAs were extracted, purified, and resolved on a PAGE gel. Radioactive signals were detected by PhosphorImager and quantified by Quantity One. ChIPs with anti-GFP and anti-Pol II were performed as described [34]. Anti-RPB2 (Abcam) and anti-GFP antibodies (Clontech) were used for IP. Enrichment of DNA fragments was measured by qPCR. The primers used in ChIP-PCR are listed in S2 Table. To test DSP1–PoII interaction, proteins were extracted from dsp1-1 harboring the GFP-DSP1 transgene. To test CPSF73-I–Pol II interactions, proteins were extracted from N. benthamiana transiently expressing GFP-CPSF73-I. To test the interactions among DSP1, DSP2, DSP4, and CPSF73-I, proteins were co-expressed in N. benthamiana. To analyze multi-protein–containing complexes, samples were treated with formaldehyde to fix protein–protein interactions as described [52]. To test the interaction of DSP3 with other proteins, a DSP3-MYC fragment was generated using primers containing elements required for in vitro transcription and translation (S2 Table). The resulting DNA fragment was used as a template to synthesize DSP3-MYC protein using a PURExpress In Vitro Protein Synthesis Kit (New England Biolabs). To obtain plants harboring two transgenes, transgenic Arabidopsis harboring GFP-DSP1 was crossed with transgenic plants containing MYC-CPSF73-I or MYC-DSP4 transgenic, whereas transgenic Arabidopsis harboring GFP-DSP4 was crossed with MYC-CPSF73-I transgenic lines. F1 plants harboring both transgenes were used for IP assay. Pollen viability was examined after Alexander’s staining [53]. In vitro pollen growth assays were performed as described [54]. To examine pollen tube growth in Col-0 and dsp1 in vivo, pistils were pollinated and collected 12 h later, then cleared and stained with decolorized aniline blue [54].
snRNAs form the RNA components of the spliceosome and are required for spliceosome formation and splicing. The generation of snRNAs involves 3′ end endonucleolytic cleavage of primary snRNA transcripts (pre-snRNAs). The factors responsible for pre-snRNA 3′ end cleavage are known in metazoans, but many of these components are missing in plants. Therefore, the proteins that catalyze pre-snRNA cleavage in plants and the mechanism leading to plant snRNA 3′ maturation are unknown. Here, we show that a DSP1 complex (containing DSP1, DSP2, DSP3, DSP4, and CPFS73-I) is responsible for pre-snRNA 3′ end cleavage in Arabidopsis. We further show that CPSF73-I, which is known to cleave the pre-mRNA 3′ end, is likely the enzyme also catalyzing snRNA 3′ end maturation in plants. Interestingly, plants appear to use two different CPSF73-I-containing complexes to catalyze the maturation of mRNAs and snRNAs. The study thereby identifies an snRNA-processing complex in plants and also elucidates a new role for CPSF73-I in this process.
biotechnology
reverse transcriptase-polymerase chain reaction
plant anatomy
protein interactions
brassica
rna extraction
pollen
plant science
model organisms
genetically modified plants
molecular biology techniques
plants
extraction techniques
genetic engineering
research and analysis methods
arabidopsis thaliana
genetically modified organisms
protein extraction
artificial gene amplification and extension
proteins
molecular biology
genetic loci
agriculture
biochemistry
plant and algal models
polymerase chain reaction
genetics
biology and life sciences
agricultural biotechnology
plant biotechnology
organisms
snRNA 3′ End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis