Heterogeneous Nuclear Ribonucleoprotein H1 Coordinateswith Phytochrome and the U1 snRNP Complex to RegulateAlternative Splicing in Physcomitrella patens[OPEN]

Chueh-Ju Shih,a,b,c Hsiang-Wen Chen,a Hsin-Yu Hsieh,a Yung-Hua Lai,a Fang-Yi Chiu,a Yu-Rong Chen,a andShih-Long Tua,b,d,1

a Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, TaiwanbMolecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Chung-Hsing University andAcademia Sinica, Taipei 11529, TaiwancGraduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, TaiwandBiotechnology Center, National Chung-Hsing University, Taichung 402, Taiwan

ORCID IDs: 0000-0002-2864-8541 (C.-J.S.); 0000-0003-2574-6127 (H.-W.C.); 0000-0001-8457-6872 (H.-Y.H.); 0000-0001-5020-400X (Y.-H.L.); 0000-0002-8664-8726 (F.-Y.C.); 0000-0002-4051-8619 (Y.-R.C.); 0000-0001-9436-278X (S.-L.T.)

Plant photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although gene expression ismodulated by photoreceptors at various levels, the regulatory mechanism at the pre-mRNA splicing step remains unclear.Alternative splicing, a widespread mechanism in eukaryotes that generates two or more mRNAs from the same pre-mRNA,is largely controlled by splicing regulators, which recruit spliceosomal components to initiate pre-mRNA splicing. The red/far-red light photoreceptor phytochrome participates in light-mediated splicing regulation, but the detailed mechanismremains unclear. Here, using protein-protein interaction analysis, we demonstrate that in the moss Physcomitrella patens,phytochrome4 physically interacts with the splicing regulator heterogeneous nuclear ribonucleoprotein H1 (PphnRNP-H1) inthe nucleus, a process dependent on red light. We show that PphnRNP-H1 is involved in red light-mediated phototropicresponses in P. patens and that it binds with higher affinity to the splicing factor pre-mRNA-processing factor39-1 (PpPRP39-1)in the presence of red light-activated phytochromes. Furthermore, PpPRP39-1 associates with the core component of U1 smallnuclear RNP in P. patens. Genome-wide analyses demonstrated the involvement of both PphnRNP-H1 and PpPRP39-1 in light-mediated splicing regulation. Our results suggest that phytochromes target the early step of spliceosome assembly viaa cascade of protein-protein interactions to control pre-mRNA splicing and photomorphogenic responses.

INTRODUCTION

Light is the most important energy source for plant growth anddevelopment. To optimize light absorption, plants have evolvedsophisticatedphotoreceptor systems tosense thequality,quantity,direction, and duration of light. Phytochromes are a major class ofphotoreceptors that mainly perceive red light (RL) and far-red light(FR) and undergo photointerconversion between Pr (the RL-absorbing form) and Pfr (the FR-absorbing form) once assem-bled with tetrapyrrole chromophores. A large portion of Pfrtranslocates to the nucleus and regulates gene expression.Phytochromes function at various levels to control gene ex-pression, such as during chromatin modification, transcription,translation, andposttranslation.Accumulatingevidencesuggeststhat alternative splicing (AS) is also regulated by phytochromes(Shikata et al., 2014; Wu et al., 2014; Xin et al., 2017). However,the detailed mechanism remains unclear.

Unlike constitutive splicing, in which typical sites for intronsplicing are recognized, AS involves the use of different splicesites to generate two or more mRNAs from the same pre-mRNA.AS often leads to the production of a premature termination co-don, reduced transcript stability, and the loss or gain of domains,thus increasing transcriptome and proteome complexity. Theregulation of AS involvesmany trans-acting factors and cis-actingelements that affect RNA-RNA, RNA-protein, and protein-proteininteractions. Most auxiliary factors, such as heterogeneous nu-clear ribonucleoproteins (hnRNPs) and Ser/Arg-rich proteins, areRNA binding proteins that regulate spliceosome activity andmodulate AS (Meyer et al., 2015). hnRNPs were first defined asspecific proteins that associate with pre-mRNA in the nucleus(Piñol-Roma et al., 1988). These proteins have multiple functions,including RNA binding, mRNA trafficking, the maintenance ofmRNA stability, and translational regulation (Dreyfuss et al., 1993;Han et al., 2010). In mammalian systems, hnRNPs associate withU1 small nuclear ribonucleoproteins (snRNPs; Wilk et al., 1991).These findings reveal the importance and potential roles ofhnRNPs in regulating pre-mRNA splicing via associations withspliceosomes.Spliceosomes, which catalyze pre-mRNA splicing, are large

ribonucleoprotein complexescomposedofcis-actingelementsofthe pre-mRNA along with snRNPs and non-snRNP splicing fac-tors (Sharp, 1994; Reddy, 2001; Zhou et al., 2002). The first step in

1 Address correspondence to tsl@gate.sinica.edu.tw.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Shih-Long Tu (tsl@gate.sinica.edu.tw).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00314

The Plant Cell, Vol. 31: 2510–2524, October 2019, www.plantcell.org ã 2019 ASPB.

spliceosome assembly is recognition of the 59 splice site by U1snRNP. The precise composition of spliceosomes in plants iscurrently unclear. Several U1 snRNP proteins, such as U1-70K,U1A, U1C, LUC7a/b, PRE-MRNA-PROCESSING FACTOR40a/b(PRP40a/b), LUC7-rl, PRP39-1 and PRP39-2, might be presentin Arabidopsis (Arabidopsis thaliana), based on comparative se-quence analysis andproteomics (WangandBrendel, 2004;Konczet al., 2012). The functions of several of these factors have beenelucidated (Simpson et al., 1995; Golovkin and Reddy, 1996;Gupta et al., 2006;Wang et al., 2007). For example, PRP39, whichwasfirst identified inyeast (Saccharomycescerevisiae), is requiredfor the formation of a U1 snRNP complex as well as the splicingprocess (Lockhart and Rymond, 1994). In plants, homologs ofPRP39 affect flowering time in Arabidopsis and the orchid Dor-itaenopsis (Wang et al., 2007; Sun et al., 2012; Kanno et al., 2017).Although PRP39 is conserved among species, whether it is in-volved in regulating pre-mRNA splicing in plants remains unclear.

We previously demonstrated that phytochromes directly par-ticipate in the regulationofAS in response toRL in themodelmossspecies Physcomitrella patens (Wu et al., 2014). Here, we providemolecular evidence thataP.patensphytochrome interactswithanhnRNP (PphnRNP-H1) in thenucleus inanRL-dependentmanner.PphnRNP-H1 is involved in RL-mediated phototropic responsesin P. patens. Genome-wide analyses further demonstrated theinvolvement of PphnRNP-H1 in light-mediated splicing regulation.PphnRNP-H1alsoshowsRL-stimulated,phytochrome-dependentbinding with the U1 spliceosomal component PpPRP39-1. Muta-tion of PpPRP39-1 in P. patens resulted in the misregulation oflight-dependent AS. Our findings strengthen the hypothesis thatphytochromes directly participate in the regulation of pre-mRNAsplicing by controlling U1 snRNP activity. Furthermore, we con-firmed that PpPRP39-1 associates with the U1 snRNP complex inP. patens via one of its components, PpU1C, and that the interactionbetween PpPRP39-1 and U1C is repressed by light. These resultssuggest that phytochromes regulate AS through hnRNPs. The

interaction betweenPphnRNP-H1, PpPRP39-1, andU1Cprovidesa molecular link by which photoreceptors control the activities ofpre-mRNA splicing machinery and modulate light responses. Ourfindingssupport thenotion thatphotoreceptor-mediated regulationof pre-mRNA splicing is evolutionarily conserved in land plants.

RESULTS

Phytochromes Interact with hnRNPs in Vitro and in Vivo

AS is differentially regulated upon RL irradiation. Photoreceptors,especially phytochromes, primarily participate in this process(Shikataet al., 2014;Wuetal., 2014;Xinetal., 2017). Toexplore thepossibility that phytochromes directly regulate splicing activity,we used protein-protein interaction analyses to test whetherphytochromes interact with any auxiliary spliceosomal factor. Inyeast two-hybrid (Y2H)-targetedscreeningof a full-length splicingregulator library, we identified an hnRNP-H-type protein that in-teracted with P. patens phytochrome4 (PpPHY4) in yeast cellsfed with phycocyanobilin (PCB), a chromophore that generatesphotoconvertible phytochromes (Supplemental Figure 1). Wenamed this protein PphnRNP-H1. In Y2H assays, yeast cellsexpressing PpPHY4 and PphnRNP-H1 were fed with PCB andgrown under different light conditions. PpPHY4, the moss or-tholog of Arabidopsis phytochrome B (PHYB), interacted withPphnRNP-H1 strongly in yeast cells grown in RL but weakly inyeast cells grown in FR or in the dark (Figure 1A). Compared withthe results from the positive (Arabidopsis PHYTOCHROMEINTERACTING FACTOR3 [AtPIF3]) and negative (PpPUBS) controls(Figure 1A), our observations indeed suggest that RL-activatedphytochromes physically interact with splicing regulators.To further explore whether photoconversion is required for

PpPHY4 to interact with PphnRNP-H1, we used the same lighttreatment as in the Y2H experiment but with the PCB analog

Phytochrome Regulates Pre-mRNA Splicing 2511

Figure 1. RL-Dependent Interaction between PpPHY4 and PphnRNP-H1.

(A) The interaction betweenPphnRNP1-H1 andPpPHY4 is enhanced byRL and in the presence of PCB. Yeast cells coexpressing PpPHY4andPphnRNP-H1 fusedwith theGAL4DNAbindingandactivationdomainsweregrownonyeastdropoutmediumsupplementedwith thechromophorePCBorPEB.Yeastcells were grown in the dark (D), RL, or FR for 3 d. AtPIF3 was used as the positive control. PpPUBS, a plastid-localized protein, was used as the negativecontrol.(B) Yeast liquid culture assay. Yeast cells were fed with chromophores and grown in the dark, RL, or FR. n5 3; error bars show the SD values. *, P < 0.01; **,P < 0.001, two-tailed Student’s t test.(C) RL-dependent interaction between PpPHY4 and PphnRNP-H1 in moss cells. Gametophore cells transiently expressing PpPHY4 and PphnRNP1-H1fused with the N- and C-terminal halves of YFP were grown in the dark (top row) and irradiated with RL for 1 h (bottom row). Nucleus-targeted mCherry(NLS-mCherry) was coexpressed as a control to visualize the nucleus. Chlorophyll autofluorescence was used to localize chloroplasts. n $ 5.

2512 The Plant Cell

phycoerythrobilin (PEB) as the chromophore to lock phytochromesin the Pr form (Li and Lagarias, 1992). Under RL, PEB-boundphytochrome interacted with PphnRNP-H1 much more weaklythan did the PCB-bound form, indicating that photoactivation isrequired for promoting protein-protein interactions between phy-tochromes and PphnRNP-H1 (Figure 1A). We quantified this in-teraction using a liquid culture assay and found that the interactionbetween PpPHY4 and PphnRNP-H1 was significantly promotedunder RL but not FR, confirming the RL-induced interaction be-tween PpPHY4 and PphnRNP-H1 (Figure 1B).

To test the interaction in vivo, we first investigated subcellularlocalization of the two proteins upon light irradiation by transientlyexpressing GFP-tagged proteins in moss cells. PphnRNP-H1 lo-calized to the nucleus regardless of light conditions (SupplementalFigure 2). PpPHY4behaved likeArabidopsis PHYB,which is presentin the cytosol in the dark and translocates to the nucleus oncethe RL signal has been received (Supplemental Figure 2). BecausePphnRNP-H1 colocalized in the nucleus with PpPHY4 upon RL ir-radiation, we further examined the RL-dependent interaction be-tweenPpPHY4andPphnRNP-H1invivobyperformingabimolecularfluorescence complementation (BiFC) assay in moss cells. Aftertransiently coexpressing PpPHY4 and PphnRNP-H1 fused with theN-terminal andC-terminal halves of yellow fluorescent protein (nYFPandcYFP, respectively), the cellswere grown in the dark orRL for 1 hand monitored for YFP fluorescence. In the dark, YFP signals werebarely observed in moss cells. However, after RL irradiation, YFPfluorescence was clearly detected in the nucleus (Figure 1C).

To more quantitatively measure the interaction between theseproteins in both the dark and light, we performed amodified BiFCassay known as ratiometric BiFC (rBiFC; Grefen and Blatt, 2012).We monitored YFP fluorescence derived from the protein-proteininteraction and normalized it to the fluorescence intensity of redfluorescent protein (RFP) constitutively expressed from the sameBiFC construct to obtain the relative fluorescence intensity(Figure 1D). The interaction between PpPHY4 and PphnRNP-H1was rapidly induced in response to RL irradiation (Figure 1E). Theinteraction between PpPHY4 and PphnRNP-H1 after RL treat-ment was also observed in the same cell when coexpressed inorchid (Phalaenopsis sogo yukidian ‘V3’) petals (SupplementalFigure 3), indicating that PpPHY4 and PphnRNP-H1 indeed in-teract in vivo. These results suggest that RL promotes the nuclearlocalization of PpPHY4 to allow for its physical interaction withPphnRNP-H1.

PphnRNP-H1 Positively Regulates Phototropic Responsesin P. patens

RL triggers phototropic responses in mosses and ferns (Coveet al., 1978; Kadota et al., 1982). Phytochromes, especially

PpPHY4, are thought to be the main photoreceptors that sensethe direction of light and control directional protonemal cellgrowth inP. patens (Mittmann et al., 2004). To investigatewhetherPphnRNP-H1 functions in photomorphogenic responses inmosses, we generated PphnRNP-H1 knockout mutants inP. patens (Supplemental Figure 4) by genome editing viaCRISPR/Cas9 and examined RL-mediated phototropic responses inthe wild type, the PpPHY4 overexpression line (PpPHY4OE;Supplemental Figure 5), and phy4 and hnrnp-h1 mutants. Weinitially grew the protonemata under negative gravitropic con-ditions in the dark. After irradiating the protonemata with 1 mmolm22 s21 unilateral RL for 1 to 4 h to induce positive phototropism,we measured the bending directions and angles of the pro-tonemata. PpPHY4OE showed increased sensitivity to the RLdirection, whereas phy4 showed reduced sensitivity, suggestingthat PpPHY4 is important for phototropism (Figure 2A). In-triguingly, hnrnp-h1 protonemata showed reduced sensitivity tounilateral RL after 1 and 4 h of RL irradiation, with a phenotypehighly similar to that of phy4, indicating that PphnRNP-H1 playsa role in photomorphogenesis.We alsomeasured the bending angles of wild-type, PpPHY4OE,

phy4, and hnrnp-h1 protonemata in response to unilateral RL(Figure 2B). The average bending angle increased during RLirradiation, but to different degrees among lines (Figure 2C;Supplemental Table 1). After a 1-h RL treatment, wild-type andPpPHY4OEprotonemata started tobend10 to20° toward the light,whereas most phy4 and hnrnp-h1 protonemata remained at anangle of 0 to 10°. The average bending angle of hnrnp-h1 pro-tonemata was also significantly smaller than that in the wild typeafter 1 h of lateral RL irradiation (Figure 2D). These findings indicatethat PphnRNP-H1 is indeed involved in regulating RL-inducedphototropic responses, especially during the early step of di-rectionsensing. ItappearsthatPphnRNP-H1functionsasapositiveregulator to fine-tune the phototropic response in P. patens.

hnRNP-H1 Functions in Light-Regulated AS

To determine whether PphnRNP-H1 is involved in light-regulatedAS, we subjected hnrnp-h1 and phy4 tomRNA sequencing (RNA-seq) to identify the changes in AS in response to RL. As illustratedin theRNA-seqflowchart (Supplemental Figure6), protonemataofwild-type,hnrnp-h1, andphy4plantsweregrown in thedark for3d(dark-grown control) and exposed to constant RL for 1 h (R1) and4 h (R4). For RNA-seq, we subjected total RNA (three biologicalreplicates per sample from independent light treatments) to librarypreparation and sequencing. After data trimming and filtering,more than 2.37 billion reads were generated (Supplemental Ta-ble 2). The sequence reads were mapped to the Physcomitrellagenome annotation V3.3 using BLAT (Kent, 2002) and Bowtie 2

Figure 1. (continued).

(D) Construct used in the rBiFC assay for PpPHY4-PphnRNP-H1 interaction. The plasmid was transformed into moss protoplast cells for transientexpression and observed by confocal microscopy.(E)Quantification of PpPHY4-PphnRNP-H1 interaction in vivo in response to RL. The radiometric BiFC construct was introduced into protoplasts from thewild type for transient expression.Regeneratedcells (darkgrown)were irradiatedwithRL for1h (R1)and4h (R4)beforeobservationbyconfocalmicroscopy.Fluorescence intensities of R1 and R4 were normalized to that of dark-grown cells. n $ 30. Data are means 6 SE.

Phytochrome Regulates Pre-mRNA Splicing 2513

(LangmeadandSalzberg, 2012).More than95%of the readswereperfectly aligned to the reference genome. We analyzed the threemajor types of AS events: intron retention (IR; Supplemental DataSet 1), exon skipping (ES; Supplemental Data Set 2), and alter-native donor and/or acceptor site (AltD/A; Supplemental DataSet 3).

We previously demonstrated that a 1-h RL irradiation causessignificantAS,especially IR (Wuetal., 2014).Wefirst examined theRL-regulated IR events in wild-type, phy4, and hnrnp-h1 pro-tonemata. Todetermine the IR level, the readcoveragedepthof anintron was divided by that of the two neighboring exons. We thencompared the three IR levels of the dark-grown and RL-irradiatedsamples (biological repeats from three independent experiments)by Student’s t test. We defined events with P # 0.005 as RL-regulated IR events. Of the 62,656 IR events identified in the wildtype, 1576 were significantly responsive to RL. To investigate theinvolvement of PpPHY4 and PphnRNP-H1 in this process, weexamined whether these events were still significant in the phy4and hnrnp-h1 mutants. Of the 1576 IR events examined, 188(12%),115 (7%),and90 (6%)eventsstill showedRL-responsive IR

in phy4, hnrnp-h1, and both lines, respectively. Intriguingly, 1183(75%) events were no longer RL responsive in either mutant, in-dicating that PpPHY4 and PphnRNP-H1 regulate the RL re-sponsiveness of these IR events (Figure 3A).To compare the RL responsiveness of these IR events, we

performed Pearson correlation analysis to categorize the eventsbased on the IR patterns in phy4 and hnrnp-1 and constructeda heatmap showing IR levels in the wild type, phy4, and hnrnp-h1(Figure 3B; Supplemental Data Set 4). Of the 1169 events thatcouldbe log2 transformed,869eventsdisplayedsimilarpatterns inphy4 and hnrnp-h1. Although the wild type also showed similarpatterns, the RL-induced changes in these events were moredramatic in the wild type than in the two mutants. As indicated inthe box plot (Figure 3C), the median fold-change values for bothRL-upregulated and -downregulated IR events (2.9 and 2.3, re-spectively) were significantly higher in the wild type than in themutants. These findings indicate that both PpPHY4 andPphnRNP-H1 play roles in modulating RL-responsive IR.We subjected the 1183 IR events to functional enrich-

ment analysis and classified them into Gene Ontology terms.

Figure 2. PphnRNP-H1 Functions in RL-Mediated Phototropism in P. patens.

(A) The percentage of protonemata in the wild type (WT), the PpPHY4 overexpression line (PpPHY4OE), phy4, and the hnrnp-h1 knockoutmutant showingpositive phototropic responses after 1 and 4 h of RL irradiation. Error bars show the SE values of three biological replicates; n of each replicate $ 50.(B) Experimental setup. Protonemata were grown vertically in the dark (top diagram) or under unilateral RL (bottom diagram). The angular histogram showsthe bending angle (u) of the protonema.(C) Angular histogram showing the bending angle (grouped in 10° intervals) and proportion (inner circle, 0%; outer circle, 100%) in wild-type, PpPHY4OE,phy4, and hnrnp-h1 protonemata in response to unilateral RL treatment for 1 and 4 h.(D)Average bending angle ofwild-type, PpPHY4OE,phy4, andhnrnp-h1protonemata in response to unilateral RL. Error bars show the SE values. n$156. *,P < 0.05; **, P < 0.001, two-tailed Student’s t test.

2514 The Plant Cell

Translation-related genes were significantly enriched among IRevents (Figure3D). This result is consistentwithourfinding that theIR patterns of translation-related genes are strongly altered aftera 1-h RL irradiation (Wu et al., 2014). These findings suggest thatPpPHY4 and PphnRNP-H1 are involved in regulating IR in thesetranslation-related genes in the light.

We selected several translation-related genes for validation byRT-qPCR. Most genes showed similar RL-dependent IR patterns

in RNA-seq and RT-qPCR analyses (Supplemental Figure 7). Innonvascular plants, phototropism is mainly controlled by phyto-chromes, and the directional growth of filamentous tip cells re-quires the rearrangement of filamentous actin (Meske andHartmann, 1995;Meske et al., 1996). As revealedbyRNA-seq andRT-qPCR, the level of IR in several actin-relatedgenes in responseto RLwas reduced in hnrnp-h1 and phy4 (Supplemental Figure 7).We also identified RL-regulated AltD/A and ES events in the wild

Figure 3. PphnRNP-H1 Is Involved in RL-Regulated IR.

(A) RL-responsive IR events in the wild type, phy4, and hnrnp-h1. The donut chart shows that out of 1576 events significantly altered in the wild type, 75%(1183) of events were not significantly regulated by RL in phy4 or hnrnp-h1. The number of events (115, 90, and 188) represents the number of events withsignificant RL-regulated IR in the wild type and mutant lines.(B)Patternsof IR levels among the1183PpPHY4-andPphnRNP-H1-dependent,RL-regulated IRevents. IR levelsafter dark (D)and1hofRL irradiation (R1)in the wild type (WT), phy4, and hnrnp-h1 lines were log2 transformed. Events with an IR level of 0 were removed from the list; 1169 events are shown in theheatmap. The eventswere grouped byPearson correlation analysis of theD toR1 pattern inphy4 and hnrnp-h1 and sorted by theR1/D fold changes inWT.The scale of fold change is shown on the bottom right side of the heatmap.(C) Box plots of upregulated (top panel) and downregulated (bottom panel) IR events. The log2 IR levels of 662 events (upregulated) and 205 events(downregulated) were plotted. The median IR level of R1 was divided by that of D to yield the fold change for upregulated events and vice versa fordownregulated events. The IR levels of each line were used to determine statistical significance by two-tailed Student’s t test. WT, wild type.(D) Functional enrichment of PpPHY4- and PphnRNP-H1-dependent IR events. The corresponding genes of the 1183 IR events were submitted to theGOBUfunctional enrichment tool, resulting in722uniquegeneswithannotations.Termswere rankedby thePvalueofoverrepresentationandwere includedin the list if P < 1E–04 (1024). BP, biological process; CC, cellular component; MF, molecular function.

Phytochrome Regulates Pre-mRNA Splicing 2515

type and compared the splicing patterns of these events amongthe wild type, phy4, and hnrnp-h1 (Supplemental Figure 8). RL-regulated AltD/A and ES events were also highly misregulated inboth mutants, further confirming the importance of PpPHY4 andPphnRNP-H1 in regulating light-mediated splicing. In summary,PphnRNP-H1 indeed regulates light-mediated splicing of specificgene transcripts to control light responses in plants.

RL Promotes the Interaction between PphnRNP-H1 and theSplicing Factor PpPRP39-1

To identify proteins that function together with PphnRNP-H1 toregulate splicing, we performed another Y2H screening of the

moss cDNA library to detect interacting proteins of PphnRNP-H1.Among the interacting proteins was the splicing-related factor,PpPRP39-1 (Supplemental Table 3). PRP39 has three putativeparalogs inP. patens, namedPpPRP39-1, -2, and -3. PpPRP39-1is evolutionarily separated from PpPRP39-2 and -3, as it sharesonly 59.3 and 57.4% similarity with these proteins, respectively(Supplemental Figure 9). PphnRNP-H1 interacted with PpPRP39-1,but not with PpPRP39-2 or PpPRP39-3, in the Y2H assay(Figure 4A), suggesting that PpPRP39-1 plays a unique role withPphnRNP-H1.Since the interaction between PpPHY4 and PphnRNP-H1 is

RL dependent, we wondered whether the interaction betweenPphnRNP-H1 and PpPRP39-1 is affected by RL as well. We

Figure 4. PpPHY4 Is Required for the RL-Promoted Interaction between PphnRNP-H1 and PpPRP39.

(A)Yeast cells coexpressingPphnRNP-H1 and three PpPRP39paralogs (PpPRP39-1, -2, and -3) fusedwith theGAL4DNAbinding and activation domainswere grown on yeast dropoutmedium (SD/–Leu/–Trp/–Ade/–His for interaction selection; SD/–Leu/–Trp for transformation selection). Empty vector (vectoronly) was used as the control.(B) The interaction of PphnRNP-H1 and PpPRP39-1 in the dark (D) or after RL treatment for 1 h (R1) and 4 h (R4) in wild-type (WT), pubs hy2, and phy4 lines.Green fluorescenceofYFP represents the interactionofPpPRP39-1 andPphnRNP-H1 (column1 from the left). Red fluorescence representsRFPsignals asthe internal control (column2). Chlorophyll autofluorescence (Chl), differential interference contrast (DIC), andmerge images are shown in columns3, 4, and5, respectively.(C)Quantification of the PphnRNP-H1-PpPRP39-1 interaction in vivo in response to RL. The rBiFC construct was introduced into protoplasts from thewildtype (WT), phytochrome-deficientmutant (pubs hy2), andphytochrome knockoutmutant (phy4) for transient expression. Regenerated cells (dark grown [D])were irradiatedwithRL for 1h (R1) and4h (R4) andobservedbyconfocalmicroscopy. Fluorescence intensities ofR1andR4werenormalizedwithD.n$30.Data are means 6 SE.

2516 The Plant Cell

performed BiFC and found that PphnRNP-H1 interacted withPpPRP39-1 in the nucleus in both the dark and RL, but the fluo-rescence intensity varied with RL irradiation level (SupplementalFigure 10). We further tested the interaction of PphnRNP-H1 andPpPRP39-1 in response to RL using rBiFC (Figure 4B). AlthoughYFP fluorescence was observed in the dark, the relative fluores-cence intensity increased in response to RL irradiation, suggestingthat RL promotes the association between PphnRNP-H1 andPpPRP39-1 (Figure 4C). Intriguingly, when we measured theinteraction in the phytochrome-deficient mutant pubs hy2 byrBiFC (Chen et al., 2012), the RL-promoted interaction betweenPphnRNP-H1 and PpPRP39-1 was lost, indicating that photo-activated phytochromes are required for the interaction ofPphnRNP-H1 and PpPRP39-1 (Figure 4C). Because PphnRNP-H1interacts with PpPHY4, we examined whether PpPHY4 affectsthe PphnRNP-H1-PpPRP39-1 interaction. The RL-promoted in-teraction between PphnRNP-H1 and PpPRP39-1 was also absentin the phy4 mutant, indicating that PpPHY4 plays a key role inthe light-mediated interaction of PphnRNP-H1 and PpPRP39-1(Figure 4C).

PRP39 Functions in Light-Regulated AS

To determine whether PpPRP39-1 is involved in regulating AS inresponse to RL, we generated the prp39-1 knockout mutant(Supplemental Figure 11), collected total RNA from light-treatedprp39-1protonema, subjected it toRNA-seq, andperformeddataanalysis (Supplemental Figure 6; Supplemental Data Set 1) todetect IR patterns for the RL-responsive events identified in thewild type in the prp39-1 mutant. Of the 1576 RL-responsiveIR events, 1331 (84.5%) were misregulated in prp39-1, clearlyindicating that PpPRP39-1 is important for RL-regulated AS(Figure 5A).We thencompared the1331eventswith those that arealso defective in phy4 and hnrnp-h1 and found that most of themoverlapped (Figure 5B). Functional enrichment analysis indicatedthat the Gene Ontology terms of these genes are highly similar tothose of PpPHY4- and PphnRNP-H1-dependent IR events(Supplemental Table 4). We validated the expression of several ofthese genes by RT-qPCR (Supplemental Figure 7). These resultsindicate that PpPRP39-1 not only functions in the regulation oflight-mediated splicing but also coordinates with PpPHY4 andPphnRNP-H1 to regulate AS of specific gene transcripts in re-sponse to RL.

PRP39 Associates with the U1 snRNP Component U1C

Prp39p is an essential component that facilitates the assembly ofthe commitment complex between U1 snRNP and pre-mRNA atthe primary step of the splicing reaction in yeast (Lockhart andRymond, 1994). Prp39p interacts with U1 snRNP and potentiallywith U2 snRNP proteins for spliceosome assembly (Gavin et al.,2002, 2006; Krogan et al., 2006; Schwer et al., 2011; Chang et al.,2012). Because the molecular function of PRP39 in plants isunknown, we investigated whether PpPRP39-1 interacts withcore components of U1 snRNP in a Y2H assay. We detectedstrong interactions between PpPRP39-1 and two U1C proteins(Figure 6A), suggesting that PpPRP39-1 comes in contactwithU1snRNP through its association with U1C.

We also observed this interaction in vivo in an rBiFC assay.Intriguingly, after RL irradiation, the interaction between PpPRP39-1and U1C decreased (Figure 6B). In phy4 cells, the RL-dependentregulation of the interaction between PpPRP39 and U1C was muchless pronounced (Figure 6B). By contrast, the association of

Figure 5. PpPRP39-1 Is Involved in RL-Regulated IR.

(A) The strategy used to analyze PRP39-1-regulated IR events. Of the1576 RL-responsive events identified in the wild type, 1331 events weremisregulated in prp39-1 and defined as PRP39-1-dependent events.(B) Venn diagram comparing IR events regulated by PRP39-1, PHY4, andhnRNP-H1.

Phytochrome Regulates Pre-mRNA Splicing 2517

PpPRP39-1 with U1C was stronger in hnrnp-h1 cells after RLtreatment (Figure6C).These resultssuggest that, unlike the inhibitoryrole of hnRNP-H1, other factors might promote the interaction be-tweenPpPRP39-1 andU1C. In summary, these results suggest thatthe association of PpPRP39 with U1C, or U1 snRNP, might bemodulated by light and potentially regulated by PphnRNP-H1.

DISCUSSION

In thisstudy,we investigatedhow lightcontrolsASviaphytochromesandsplicing regulators.We identifiedasplicing factor,PphnRNP-H1,which plays important roles in RL signaling to regulate splicing. Inaddition to interacting with phytochrome, PphnRNP-H1 associateswith the U1 snRNP component PpPRP39-1, perhaps to modulatesplicing activity. PpPRP39-1 might serve as an auxiliary factor of U1snRNP in plants, acting as the bridge that mediates signaling fromphytochromes to spliceosomes, thereby regulating light-responsiveAS. Based on these and previous findings, we propose a modeldescribing how phytochromes regulate AS via PphnRNP-H1 andPpPRP39-1 under different light conditions (Figure 7). In the dark,phytochromes (Pr) are mainly localized to the cytoplasm, whereas in

the nucleus, the interaction between PphnRNP-H1 and PpPRP39-1occurs at a basal level to modulate U1 snRNP activity. After plant-s perceive light, photoactivated phytochromes translocate to thenucleus and interact with PphnRNP-H1, which in turn promotesPphnRNP-H1’s interaction with PpPRP39-1. The enhancedinteraction between PphnRNP-H1 and PpPRP39-1 might causePpPRP39-1 to dissociate from the U1 snRNP component U1C,thereby controlling splicing.U1 snRNP is the first RNP complex to associate with the 59 splice

site (59ss) of pre-mRNA and promote the stepwise assembly of theother components that form the active spliceosome (Will andLührmann, 2011). Therefore, the proper formation of the U1 snRNPcomplex is crucial for spliceosome function. U1-specific proteins,such as PRP39, are critical for the formation and stability of the U1snRNP-pre-mRNA complex (Lockhart and Rymond, 1994; McLeanand Rymond, 1998; Zhang and Rosbash, 1999). The Arabidopsisprp39 mutant exhibits preferential splicing of the U2-type AT-ACintron (Wang et al., 2007; Kanno et al., 2017), suggesting that plantPRP39 might also function in splice site recognition by U1 snRNP.U1C stabilizes the binding between U1 snRNP and the 59ss(Heinrichs et al., 1990). Du and Rosbash (2002) demonstrated that

Figure 6. Interaction between PpPRP39-1 and Spliceosomal Components.

(A)Yeast cells coexpressingPRP39 andP. patensU1A (PpU1A), two paralogs of U1C (PpU1C-1 and -2), and two paralogs of U1-70K (PpU1-70K-1 and -2)fused with the GAL4 DNA binding and activation domains were grown on yeast dropout medium (SD/–Leu/–Trp/–Ade/–His for interaction selection;SD/–Leu/–Trp for transformation selection). Empty vector was used for the control.(B) to (D)Quantification of rBiFC results to determine the interaction between PpPRP39-1 and PpU1C after RL irradiation in thewild type (B), phy4 (C), andhnrnp-h1 (D). Moss protoplasts transiently expressing PpPRP39-1 and PpU1C fusedwith the N- and C-terminal halves of YFPwere observed by confocalmicroscopy. RFP was coexpressed as an internal control. Chlorophyll autofluorescence was used to localize chloroplasts. Regenerated cells (dark grown[D]) were irradiated with RL for 1 h (R1) and 4 h (R4) prior to observation by confocal microscopy. Fluorescence intensities of R1 and R4were normalized tothat of D. n $ 39. Data are means 6 SE. Data were used to determine statistical significance by two-tailed Student’s t test. *, P < 0.005; **, P < 0.001.

2518 The Plant Cell

U1Cbindsdirectly to the 59ss in yeast. Althougha recently publishedhigh-quality crystal structure indicates that human U1 snRNP bindsdirectly tothe59ssofpre-mRNAviaU1-70K,U1Cmightfine-tuneandstabilize this binding (Kondo et al., 2015). Perhaps U1C functions asthe entry site on plant U1 snRNP to control the recognition andbinding affinity of this complex to the 59ss. In this study, we dem-onstrated that PpPRP39-1, together with PpPHY4 and PphnRNP-H1, regulates a majority of RL-responsive IR (Figure 5). Our rBiFCexperiments indicated that RL promotes the interaction betweenPphnRNP-H1 and PpPRP39-1 (Figure 4) but inhibits the associationof PpPRP39-1 with U1C (Figure 6). Perhaps light activates thesplicing repressor hnRNP, allowing it to occupy PRP39 and pre-venting it from associating with U1C. This would lead to a defect insplice site recognitionbyU1snRNP, resulting in light-responsiveAS.

Compared with the extensively characterized Ser/Arg-rich pro-teins (Reddy and Shad Ali, 2011), our understanding of the functionof hnRNPs in plants is still limited. Information about plant hnRNPshas primarily been obtained from studies of three types of hnRNPsin Arabidopsis: polypyrimidine tract binding proteins, GLYCINERICH PROTEIN7 (GRP7), and GRP8 (Staiger et al., 2003; Schöninget al., 2008; Stauffer et al., 2010). In this study, we provide evidencethat an H-type hnRNP physically interacts with phytochrome uponlight irradiation (Figure 1). The strong similarity of the phototropicresponsesofhnrnp-h1andphy4also indicates thatPphnRNP-H1 isinvolved in PpPHY4-regulated phototropism (Figure 2). Further-more, themisregulation of AS after RL treatment in phy4 and hnrnp-h1 suggests that PpPHY4 and PphnRNP-H1 both function in theregulation of IR (Figure 3). These findings strongly support thefunctional importance of hnRNP in the light-signaling pathway.

In terms of splicing regulators in the light-signaling pathway, anortholog of the human potential splicing factor SR140, REDUCED

RED-LIGHT RESPONSES IN CRY1CRY2 BACKGROUND1, isthought to be important for phytochrome B signaling in Arabidopsis(Shikata et al., 2012). In this study, we demonstrated that thehnRNP,PphnRNP-H1, interactswithphytochromeandtakespart inphotoreceptor-mediated splicing regulation in response to light. An-other recentstudyshowedthat themutationofSPLICINGFACTOR45,a potential U2 snRNP component, results in photomorphogenic de-fects includingearlyflowering (Xinetal.,2017).Together, thesefindingsstrongly suggest that prespliceosome (complex A) formation andactivity are regulated by light. Although it is still not clear how theprespliceosome determines transcript specificity during the light-mediated regulation of pre-mRNA splicing, auxiliary factors such asPRP39, which help bridge U1 and U2 snRNPs for prespliceosomeassembly, could be the targets of light-activated splicing regulatorssuch as hnRNP-H1 to modulate prespliceosome activity.Although photoreceptor-independent processes such as plastid-

to-nucleus signaling and changes in energy availability potentiallyregulate AS (Petrillo et al., 2014;Hartmann et al., 2016;Mancini et al.,2016), there is unequivocal evidence for the requirement of phyto-chromes in light-regulated AS (Shikata et al., 2014; Wu et al., 2014;Xin et al., 2017). Both pathways could be present in plant cells.Photoreceptor-mediated regulation, which is more sensitive, couldbe the primary event that occurs immediately after light exposure.Plastid signaling might be induced after longer light exposure andunder higher light intensity because they require the saturation ofphotosyntheticreactions.Bothpathwayscouldcoexist toprovidethemultilayer regulation of gene expression. We believe that multiplesplicing regulators are involved in light-dependent regulation for theproduction of alternatively spliced transcripts. Crosstalk betweenthese factors and spliceosomal components might occur in a co-ordinatedmanner to produce the transcripts necessary for important

Figure 7. Molecular Model of Phytochrome-Regulated AS.

According to thismodel, in thedark, phytochromes aremainly found in the inactive form in the cytosol. LessPphnRNP-H1 interactswithPpPRP39-1,whichstably associates with PpU1C on U1 snRNP. In the light, photoactivated PpPHY4 translocates to the nucleus and interacts with the splicing regulatorPphnRNP-H1. Phytochromes also promote the association of PphnRNP-H1 andPpPRP39with higher affinity. PpPRP39dissociates fromU1snRNPuponRL irradiation, which may reduce the activity of U1 snRNP, resulting in AS.

Phytochrome Regulates Pre-mRNA Splicing 2519

biological activities such asdevelopmental programsand responsesto environmental cues. Identifying other components involved in thisregulatory network and uncovering their functions are importantareas for further investigation.

AS of individual regulatory genes could potentially affect plantgrowth anddevelopment; however, the global pattern of AS in genessuch as those involved in metabolism can have an even strongerinfluence on plant growth. Because AS greatly increases tran-scriptome complexity and proteome diversity, it provides an addi-tional layer of gene regulation to reprogrambiological activities in thecell. This mechanism likely plays an important role in plants underchanging environmental conditions because posttranscriptionalregulation allows for immediate responses to dynamic conditions.Our findings provide insights into how AS is modulated in plants viathe coordinated activities of photoreceptors, splicing regulators, andthe spliceosome in response to light conditions. More data areneeded to provide additional details about this mechanism, such asthe involvementof other splicing regulatorsor spliceosomal proteins,in order to obtain a complete view of this complicated network.Furthermore, it would be worthwhile to explore the participation ofother photoreceptors and their strategies for regulatingAS inorder tocompletely understand the mechanism that regulates AS.

METHODS

Plant Materials and Growth Conditions

Protonemata of Physcomitrella patens subsp patens were grown on solidBCDAT medium, and gametophores were grown on Knop’s medium.Spores were germinated on cellophane-overlaid solid Knop’s mediumsupplemented with 10 mM CaCl2 and grown for 2 weeks. The plants werecultured at 24°C under continuous white light (80–100 mmol m22 s21).

Plasmid Construction

Thevectorsused forY2Handmoss transformationwereconstructedusingan In-Fusion HD Cloning kit (Clontech Laboratories). In brief, cDNA se-quences of the desired genes were amplified using primers containing 15nucleotides identical to the gene regions and 15-nucleotide extensionshomologous to the vector ends. PCR-generated sequences were as-sembled with linearized vectors using In-Fusion Enzyme and transformedinto Escherichia coli for plasmid amplification. The primers used in thisstudy are listed in Supplemental Data Set 5.

To produce the rBiFC constructs, 2-in-1 BiFC vectors were used toconstruct the plasmid containing the coding regions of PpPRP39-1,PphnRNP-H1,PpU1C, andPpPHY4byGatewaycloning (GrefenandBlatt,2012). Coding sequences ofPpPRP39-1orPpPHY4with attB3/2 sites andcoding sequences of PphnRNP-H1 or PpU1C with attB1/4 sites wereamplified and introduced into pDONR221P3P2 and pDONR221P1P4usingBP-Clonase II (ThermoFisherScientific) to generate the entry clones.The successful entry clones were incubated with pBIFCt-2in1-NN desti-nation vectors as well as LR-Clonase II (Thermo Fisher Scientific). Thereaction products were transformed into E. coli cells for plasmid amplifi-cation and selection.

Transformation and Generation of P. patens Knockout orOverexpression Lines

The hnrnp-h1 knockout line was generated using the CRISPR-Cas9system as previously described (Collonnier et al., 2017). The guide RNAsequence was designed using the CRISPOR program (Haeussler et al.,

2016). A fragment containing the 20-nucleotide guide RNA sequencedrivenby theP.patensU6promoterwascloned into thepDONR207 vectorby Gateway cloning to obtain the Pp-SpgRNA-U6 plasmid. A mixture of7 mg of Pp-SpgRNA-U6 plasmid, 7 mg of pAct-CAS9 (encoding Cas9nuclease), and 7 mg of pBNRF (provides resistance to G418) was trans-formed into P. patens protoplasts by polyethylene glycol (PEG)-mediatedtransformation (Nishiyamaet al., 2000). Fragmentscontaining themutationsite were amplified from genomic DNA obtained from 50 transformantsshowing G418 resistance and sequenced to verify the mutation. A mutantline with an 8-bp deletion at the guide RNA target site of the PphnRNP-H1locus and in the encoding mRNA was selected for further study(Supplemental Figure 4). To generate the prp39-1 knockout line, the up-streamanddownstreamregionsof thePpPRP39-1 locuswereamplifiedbyPCR and cloned into the pTN80 vector containing an nptII cassette. Togenerate the PpPHY4 overexpression line, the coding sequence ofPpPHY4 was amplified by PCR and cloned into the pTN80 vector con-taining an aph4 cassette and two fragments from an intergenic region ofP. patens chromosome 2 as the downstream and upstream sequencesfor homologous recombination. PEG-mediated transformation was per-formed as described, with minor modifications (Nishiyama et al., 2000).Seven-day-old protonemata generated from sporophytes were collectedfor protoplast isolation and PEG transformation. The transformants wereselected on BCDAT medium containing 20 mg/L G418 or 30 mg/L hy-gromycin, and gene-specific insertion in stable transformants was verifiedby PCR using specific primers to confirm gene targeting.

Y2H Analysis

The coding regions of hnRNP and PUBS from P. patens and PIF3 fromArabidopsis (Arabidopsis thaliana) were cloned into the pGEM-T vector andsubcloned into the pGADT7 vector. The coding regions of P. patens phy-tochrome genes were cloned into the pGBKT7 vector. The plasmids weretransformed into yeast (Saccharomyces cerevisiae) strainY187 (Clontech) orY2HGold (Clontech), cultured overnight in liquid YPDAmedium (blended ofyeast extract, peptone, dextrose, and adenine hemisulfate), and used fortransformation according to the manufacturer’s instruction. Two singlecolonies grown fromSD-Leu or SD-Trpmediumwere mated on solid YPDAplates for 24 h, transferred to SD/–Leu/–Trp medium, and grown for 3 d.Single transformantswere cultured for 3 d on solid SD/–Ade/–His/–Leu/–Trpmediumwith 5 mMPCB or PEB in the dark, RL (660-nm light emitting diode[LED], 5 mmol m22 s21), or FR (730-nm LED, 1 mmol m22 s21). For the yeastliquidcultureassay, thecultureswere fedwith5mMPCBorPEBandgrown inthe dark, RL (660-nmLED, 5 mmolm22 s21), or FR (730-nmLED, 1 mmolm22

s21) until the cells were in the mid-log phase. The cells were harvested, andequal amounts were subjected to a b-galactosidase activity assay usingchlorophenol red-b-D-galactopyranoside (Roche) as the substrate ac-cording to the manufacturer’s instructions (Clontech).

AP.patenscDNA library forY2Hscreeningwasconstructedaccording tothe manufacturer’s instructions (Clontech). Light- and dark-grown 7-d-oldmoss protonemata were harvested for total mRNA extraction (GMbiolab),and2mgofmRNAwasusedtogeneratecDNAswithendshomologoustothecloningsitesof thepGADT7-Recvector.Thesingle-strandedcDNApoolwasamplified by long-distance PCR. Double-stranded products were purifiedusing a CHROMA SPIN TE-400 column to select for DNA molecules longerthan 200 bp. After purification, the double-stranded cDNA and linearizedpGADT7-Rec were cotransformed into competent Y187 yeast cells. Thetransformed cells were grown on SD/-Leu medium at 30°C for 4 d. Trans-formed colonies were collected and stored at 280°C.

To identify the interacting proteins of hnRNP1, Y2H screening wasperformed using the newly constructed cDNA library. Yeast cells carryingBD-hnRNP1 (hnRNP1 constructed with a binding domain) and theAD-library (cDNA library constructed with an activation domain) werecombined in 23 YPDA medium for mating. After 20 h of incubation,the mated culture was plated on SD/-Leu/-Trp/X-a-gal/aureobasidin A

2520 The Plant Cell

medium and grown for 5 d. Blue colonies were selected and transferred toSD/-Leu/-Trp/-Ade/-His/X-a-gal/aureobasidin A plates. Colonies surviv-ing in final selectionmediumwere analyzed by yeast colony PCR and DNAsequencing.

BiFC

Particle bombardment was performed using the Biolistic PDS-1000/HeParticle Delivery System (Bio-Rad) to transiently coexpress the targetproteins fused with N- or C-split YFP in moss gametophores or orchidpetals. Plasmids containing NLS-mCherry were also included as a nuclearlocalization marker along with the other plasmids. Moss cells were re-covered overnight in the dark after bombardment and irradiated with RL(660-nm LED, 5 mmol m22 s21) for 1 and 4 h. Fluorescence was visualizedby confocal microscopy. Plant cells were observed under a LSM510 mi-croscope (Zeiss) andaLSM880microscopewithAiryscan (Zeiss) under thefollowing conditions: YFP and RFP were excited by the 514-nm line of anargon laser and the 543-nm line of a HeNe laser, respectively. YFP fluo-rescence was detected using a 535- to 560-nm band-pass filter. RFPfluorescence was detected using a 565- to 615-nm band-pass filter, andchloroplast autofluorescence was detected at 650 to 710 nm.

rBiFC

Moss protoplast preparation and transient expression via PEG trans-formation were performed as described in Physcobase (http://moss.nibb.ac.jp/). After transforming 30 mg of circular plasmid DNA into fresh pro-toplasts andanovernight recovery in thedark, theprotoplastsweredividedinto aliquots in microcentrifuge tubes and treated with RL (660-nm LED,5mmolm22 s21) for 1and4h.Cellswereobservedbyconfocalmicroscopy.At least 25 protoplasts with RFP signals were imaged for further quanti-fication. ImageJwas used to quantify YFP andRFP fluorescence intensity.Relative fluorescence intensity was calculated as YFP fluorescence in-tensity divided by the intensity of RFP (Grefen and Blatt, 2012).

Phototropism Assay

The phototropism assay was performed as described with minor mod-ifications (Mittmann et al., 2004). P. patens protonemata were grown onBCDAT with 1.5% agar (w/v) under continuous white light. The pro-tonemata were collected and transferred to vertically placed Petri dishes,sandwiched between two layers of cellophane, and incubated in the darkfor 3 d to induce caulonema formation. In the dark, caulonema filamentsgrow vertically in response to negative gravitropism. To analyze photot-ropism, dark-incubated protonemata were treated with lateral mono-chromatic RL (660-nm LED, 1 mmol m22 s21). The protonemata werephotographed under a Lumar V12 stereomicroscope (Zeiss), and thebending angle was calculated with ImageJ. The average bending angleplus SE of dark-incubated plants was defined as straight, and protonematawith larger bending angles than those of dark-incubated plants werecategorized as exhibiting positive phototropism.

Light Treatment for RNA-Seq Analysis

Moss gametophore tissues were ground with a Polytron tissue homog-enizer to regenerate protonema tissues. The homogenized plant culturewasgrown inwhite light for 7d,andprotonemacolonieswerecollectedandground with a Polytron tissue homogenizer. Homogenized protonematissue was grown in white light for another 10 d. Ten-day-old wild-typeandmutant protonemata were grown in the dark for 3 d, followed by 1- and4-h RL irradiation (660-nm LED, 5 mmol m22 s21) at 25°C as previouslydescribed (Chen et al., 2012). Dark-grown protonema cells were collectedas the dark control.

RNA Isolation and RT-qPCR Analysis

RNAwas isolated as described (Chen et al., 2012). cDNAwas synthesizedfrom 1 mg of total RNA with an oligo(dT) and SuperScript III RT kit (In-vitrogen). RT-qPCR analysis was performed using the QuantStudio 12KFlex Real-Time PCR System (Thermo Fisher Scientific) with Power SYBRGreen PCR Master Mix (Thermo Fisher Scientific), and reactions were runfor 40 cycles. Primers were designed using Primer Express v3.0 (AppliedBiosystems) and are listed in Supplemental Data Set 5. RT-qPCR wasperformed in triplicate. PpACT2 (Pp3c3_33440V3) was used as an internalcontrol for normalization.

RNA-Seq and Data Analysis

Ten-day-old wild-type and mutant protonemata were grown in the darkfor 3 d, followedby1h (R1) ofRL treatment (660-nmLED, 5mmolm22 s21)at 25°C as previously described by Chen et al. (2012). Dark-grownprotonema cells were collected as the dark control. RNA isolation andlibrary construction of three biological replicates sampled at differenttimes were performed independently as described previously by Chenet al. (2012). Sequencing was performed on the HiSeq 4000 platform atYourgene Bioscience. On average, 95million 100-nucleotide paired-endreads were obtained per library. Sequence reads were mapped to thePhyscomitrella genomeannotationV3.3 (JGI, https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias5Org_Ppatens) using BLAT (Kent, 2002)and Bowtie 2 (Langmead and Salzberg, 2012). More than 95% of thereads were perfectly aligned to the reference genome. The three majortypes of AS events, IR, AltD/A, and ES, were analyzed as describedpreviously withminormodifications (http://rackj.sourceforge.net; Kannoet al., 2017). In brief, IR was measured by comparing IR levels in the darkcontrols (biological repeats from three independent experiments) withthose in the R1 replicates using Student’s t test, where the IR level wasdefined as the average read coverage depth of the intron divided by theaverage read coverage depth of the neighboring exons. A significant Pvalue from the t test indicates that the IR level was altered in oneof the twosamples. These IR events were filtered out using the following criteria: noreplicates in two samples with 100% read coverage for the retainedintron; no replicates in twosampleswith average readcoveragedepth>1for the retained intron. RL-regulated IR events were considered to be RL-responsive when an event had P # 0.005.

ESandAltD/A eventsweredeterminedusing amethodsimilar to that forIR events. For ES events, log ratios of ES events in the dark controls werecompared with those in the R1 replicates using t tests, where the ES logratiowasdefinedas the log of the splice read count supporting anESeventdivided by the splice read count aligned to the skipped exon. For AltD/Aevents, thecorresponding log ratiowasdefinedas the logof the splice readcount supporting a splicing junction divided by the splice read countsupportingall other junctionsof thesame intron.BothESandAltD/Aeventswere further confirmed using additional t tests with log ratios of supportingread counts and unique read counts of individual genes. ES or AtlD/Aevents were filtered out using the following criteria: the sum of the splicereadcount in twosamplessupportinganESorAltD/Aevent is$10; thesumof the splice read count aligned to the skipped exon is$10 (for ES); the sumof the splice read count supporting all other junctions of the same intron is$10 (for AltD/A). Finally, an RL-regulated ES or AltD/A event was defined ifits P valueswere both<0.05. Todetermine the fold changes of ESorAltD/Alevels in response to RL, the mean ES or AltD/A ratios for R1 or R4 weredivided by those of the dark samples.

Accession Numbers

RNA-seq data from this article have been submitted to the National Centerfor Biotechnology Information Sequence Read Archive database (http://www.ncbi.nlm.nih.gov/sra) and assigned the identifier SRP146733. Gene

Phytochrome Regulates Pre-mRNA Splicing 2521

informationdescribed in thisarticle canbe found in thePhytozomedatabaseof JGI (http://www.phytozome.net/physcomitrella.php) under the followinggene locusnumbers:PphnRNP-H1 (Pp3c7_8760),PpPHY4 (Pp3c27_7830),PpPRP39-1 (Pp3c27_7380), PpU1A (Pp3c7_10880), PpU1C-1 (Pp3c12_26180), PpU1C-2 (Pp3c4_170), PpU1-70K-1 (Pp3c7_6390), PpU1-70K-2(Pp3c11_26490), PpPHY2 (Pp3c16_20280), PpVIP1/Asp1 (Pp3c3_34460),PpNPH3 (Pp3c10_19380), PpCAPZB (Pp3c1_32040), PpRPS21 (Pp3c12_25030), PpRPS6 (Pp3c15_25430), and PpRPL32 (Pp3c17_13590).

Supplemental Data

Supplemental Figure 1. Y2H screening for interactions betweenphytochromes and splicing regulators in P. patens.

Supplemental Figure 2. Subcellular localization of PpPHY4 andPphnRNP-H1 in moss cells.

Supplemental Figure 3. Red-light-dependent interaction betweenPpPHY4 and PphnRNP-H1 in the same orchid petal cell.

Supplemental Figure 4. CRISPR/Cas9 strategy and validation togenerate the hnrnp-h1 knockout line.

Supplemental Figure 5. Construction and gene targeting strategy forthe phytochrome 4 overexpression line (PHY4OE).

Supplemental Figure 6. Flowchart for analyzing the RNA-seq data.

Supplemental Figure 7. Validation of RNA-seq data.

Supplemental Figure 8. PphnRNP-H1 is involved in regulating RL-mediated AS.

Supplemental Figure 9. Sequence alignment of P. patens PRP39paralogs.

Supplemental Figure 10. Interaction between PphnRNP-H1 andPpPRP39-1.

Supplemental Figure 11. Construction and verification of gene-targeted prp39-1 mutant lines.

Supplemental Table 1. Bending angles of tip cells in the phototropismexperiment.

Supplemental Table 2. Mapping statistics of RNA sequencing.

Supplemental Table 3. PphnRNP-H1-interacting proteins identifiedby Y2H screening.

Supplemental Table 4. Functional enrichment of IR genes regulatedby PpPHY4-, PphnRNP-H1- and PpPRP39-1.

Supplemental Data Set 1. List of light-regulated IR events in WT,phy4, hnRNP-h1, and prp39-1.

Supplemental Data Set 2. List of light-regulated ES events in WT,phy4, and hnRNP-h1.

Supplemental Data Set 3. List of light-regulated AltD/A events in WT,phy4, and hnRNP-h1.

Supplemental Data Set 4. AS levels in WT, phy4, and hnRNP-h1 usedto construct the heatmaps.

Supplemental Data Set 5. List of primers used in this study.

ACKNOWLEDGMENTS

We thank Shu-HsingWu for critically reading the article and Shu-JenChouat the Genomic Technology Core Laboratory, Mei-Jane Fang at the LiveCell Imaging Core Laboratory, andWen-Dar Lin at the Bioinformatics CoreLaboratory of the Institute of Plant andMicrobial Biology, AcademiaSinica,

for technical assistance. We also thank Fabien Nogué, Institut Jean-PierreBourgin, INRA Centre de Versailles-Grignon, France, for providing theCRISPR/Cas9vectors. Thisworkwassupportedby theMinistry ofScienceand Technology, Taiwan (grant MOST 106-2311-B-001-033-MY3 toS.-L.T.) and by the Academia Sinica

AUTHOR CONTRIBUTIONS

S.-L.T. and C.-J.S. participated in the design of the study; C.-J.S. andH.-W.C. performed Y2H and BiFC experiments; C.-J.S. conducted pho-totropism analysis; C.-J.S. performed RNA extraction, mRNA sequencing,and validation experiments; S.-L.T., H.-Y.H., and C.-J.S. conducted se-quencing data analysis; C.-J.S. and F.-Y.C. generated the PHY4 over-expression line; Y.-H.L. performed the CRISPR/Cas9 experiments togenerate the hnrnp-h1 mutant; H.-W.C. carried out prp39-1 generation;Y.-R.C. constructed thesplicing regulator library forY2Hscreening;C.-J.S.and S.-L.T. wrote the article; S.-L.T. coordinated the project.

Received April 29, 2019; revised July 15, 2019; accepted August 6, 2019;published August 13, 2019.

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2524 The Plant Cell

DOI 10.1105/tpc.19.00314; originally published online August 13, 2019; 2019;31;2510-2524Plant CellShih-Long Tu

Chueh-Ju Shih, Hsiang-Wen Chen, Hsin-Yu Hsieh, Yung-Hua Lai, Fang-Yi Chiu, Yu-Rong Chen andPhyscomitrella patensComplex to Regulate Alternative Splicing in

Heterogeneous Nuclear Ribonucleoprotein H1 Coordinates with Phytochrome and the U1 snRNP

 This information is current as of December 29, 2020

 

Supplemental Data /content/suppl/2019/11/01/tpc.19.00314.DC2.html /content/suppl/2019/08/14/tpc.19.00314.DC1.html

References /content/31/10/2510.full.html#ref-list-1

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