Alternative splicing (AS) can significantly impact the transcriptome and proteome of a eukaryotic cell. Here, using transcriptome and proteome profiling data, we analyzed AS in two life forms of the model moss Physcomitrella patens, namely protonemata and gametophores, as well as in protoplasts. We identified 12 043 genes subject to alternative splicing and analyzed the extent to which AS contributes to proteome diversity. We could distinguish a few examples that unambiguously indicated the presence of two or more splice isoforms from the same locus at the proteomic level. Our results indicate that alternative isoforms have a small effect on proteome diversity. We also revealed that mRNAs and pre-mRNAs have thousands of complementary binding sites for long non-coding RNAs (lncRNAs) that may lead to potential interactions in transcriptome. This finding points to an additional level of gene expression and AS regulation by non-coding transcripts in Physcomitrella patens. Among the differentially expressed and spliced genes we found serine/arginine-rich (SR) genes, which are known to regulate AS in cells. We found that treatment with abscisic (ABA) and methyl jasmonic acids (MeJA) led to an isoform-specific response and suggested that ABA in gametophores and MeJA in protoplasts regulate AS and the transcription of SR genes.
Alternative splicing (AS) is an essential mechanism of post-transcriptional modification responsible for the transcriptome plasticity and proteome diversity of a cell. AS plays important roles in regulating many physiological processes in plants, such as plant growth and development, circadian clock function, flowering, and stress responses1,2,3,4. According to recent studies, 61% of genes are subjected to AS in Arabidopsis thaliana, whereas approximately 40% are in maize, and 33% are in rice5,6,7,8,9. The number of alternative isoforms is known to increase under plant stress10,11,12,13.
Under stress conditions, splicing of genes involved in responses to abiotic stress and RNA processing changes13, 14, thus supporting the idea that AS plays a regulatory role in stress responses15. However, the molecular mechanisms of AS regulation are poorly understood.
It is known that special regulatory proteins, such as serine/arginine-rich proteins (SR proteins) and heterogenic ribonucleoproteins (hnRNP), are involved in AS tuning. These proteins bind to specific nucleotide motifs16, 17. SR proteins are a family of highly conserved phosphoproteins that can bind directly to alternatively spliced introns18,19,20. Several studies have demonstrated that the genes encoding SR splicing factors tend to change their splicing profiles under stress conditions, suggesting a complex loop in splicing regulation15, 21. However, it is not clear whether SR proteins are subject to similar regulation in non-vascular plants.
Long non-coding RNAs are potentially important yet poorly studied AS regulators involved in many regulatory cellular processes22,23,24. They can regulate splicing in humans via lncRNA–pre-mRNA interactions25, 26, which may mask splice sites and prevent spliceosome assembly, or cause RNA to be edited via the adenosine-to-inosine (A-to-I) mechanism, resulting in splice recognition site modification24. For example, up to 50% of human genes are transcribed with their natural antisense transcripts (NATs)27, that might be associated with AS regulation28. Wang et al. identified 37 238 antisense transcripts in A. thaliana, making up approximately 70% of annotated mRNAs29; however, data on such interactions in plants are scarce.
Over 95% of mammalian multi-exon protein-coding genes have more than a single isoform30, 31; therefore, many studies have sought to identify RNA molecules generated by AS, and deduce their influence on cell proteomes32,33,34. However, knowledge on the impact of different splicing variants on plant proteomes is limited. Early comparative bioinformatics analysis has suggested a limited role for AS in plants in terms of functional expansion of the proteome35,36,37; only a small number of AS events have conserved effects at the protein sequence level in Arabidopsis and rice37. Identifying isoform-specific proteins and peptides is challenging because of the dynamic nature of the proteome, the often low abundance of splice variant-specific proteins and peptides, and the similarity of protein isoform sequences38, 39. Moreover, AS affects both the level of protein produced by a gene and the transcript stability, making it difficult to predict the coding potential of isoforms40. For example, in A. thaliana, approximately 18% genes generate isoforms that degrade via nonsense-mediated decay (NMD) caused by a premature termination codon (PTC)41, 42. In humans, 85% of mRNAs studied are predominantly transcribed as a single, major isoform43. A combination of mass spectrometry and transcription profile analysis can help elucidate the roles of AS in gene expression regulation creating proteome diversity.
The moss Physcomitrella patens is a common model organism in systems biology44,45,46. Moss has two life forms – filamentous protonemata representing the juvenile stage, and leafy gametophores representing the adult stage. Protonema filaments are used as a source of protoplasts, which are an ideal system for developmental studies because of their ability to regenerate intact plants at high frequency47. Approximately 50% of P. patens protonema genes are alternatively spliced, with the most frequent type of AS being intron retention48, 49. Stress factors and environmental conditions, such as exposure to light, both influence AS48, 49. However, the role of AS in the developmental transition from protonema to gametophore is poorly studied, as is its effect on reprogramming during protoplast isolation. There are also no data on the influence of AS on the moss proteome.
In this study, we used RNA-seq, mass spectrometry, Reverse Transcription-PCR (RT-PCR), and quantitative Reverse Transcription PCR (qRT-PCR) to investigate dynamics in the AS of genes in P. patens, and the influences of AS on the cell proteome during the transition from protonemata to gametophores or protoplasts. We found that the transcriptomes of gametophores, protonemata, and protoplasts differ considerably. During the developmental transition from protonemata to gametophores, changes in AS were associated with genes involved in carbohydrate and lipid metabolism, growth and development processes, and stress responses. In protoplasts, changes in AS were observed in genes related to transcription regulation, development, cell interactions, and cell response to biotic and abiotic stresses. Using mass spectrometry, we identified 1458 isoform-specific tryptic peptides, representing 616 alternatively spliced genes, thus confirming their translation.
To evaluate the factors regulating AS processes, we analyzed SR proteins and lncRNAs in P. patens protonemata, gametophores and protoplasts. We identified new SR gene isoforms at the transcriptome and proteome levels, and analyzed isoform-specific transcription under treatment with abscisic and jasmonic acids. We also investigated the lncRNA–mRNA and lncRNA–pre-mRNA interactions in gametophores, protonemata, and protoplasts using the identified non-coding transcripts in these cell types, and data from CANTATAdb50. Our results indicate that such interactions are important in AS regulation.
Transcriptome Profiling of Protonema, Gametophore, and Protoplast Cells Reveals an Extended Catalog of Differentially Expressed Genes
To explore the transcriptomes of juvenile and mature moss life forms, and that of protoplasts, and discover which genes are differentially expressed (DE) and differentially alternatively spliced (DAS) during developmental transition or protoplastation, we used previously described data from strand-specific poly-A RNA sequenced by ABI SOLID 4.051 as shown in Fig. 1A.
We discovered 23 446 transcribed genes reflecting 47 976 mRNA isoforms. Comparing gametophores and protonemata, 3772 DE genes were identified; 1906 were upregulated in gametophores and 1866 were downregulated in protonemata. Comparative analysis of protonema and protoplast transcriptomes revealed 1913 DE genes, with 1470 of these upregulated in protoplasts. We compared the transcription level of DE genes in protonemata, gametophores, and protoplasts using k-medians clustering (Supplementary Figure S1; Supplementary Table S1). In gametophores, genes involved in morphogenesis, growth, gametophore development, sexual reproduction, polysaccharide synthesis, and stress response were upregulated (see Supplementary Data S1). In protonemata, transcript accumulation was increased in genes involved in cell wall synthesis, photosynthetic processes, carbohydrate biosynthesis, cellular morphogenesis, cell size regulation, cell generation, cell cycle processes, and cytokinesis (Supplementary Figure S1; Supplementary Table S1).
Identification of Alternatively Spliced Genes
The most abundant types of AS in P. patens are alternate donor and/or acceptor site and intron retention; the latter accounts for 40% of splicing in moss48, 49. According to our RNA-seq data, 12 043 genes had more than one isoform, suggesting that AS had taken place; 4720 of these demonstrated intron retention splicing (Fig. 1B). Of all AS variants revealed, 57.06% were previously annotated and 42.94% identified as new splice variants. Genes with isoform transcription levels that changed by more than 20% (dIF >20, see Materials and Methods for details) were selected as DAS genes for further definition to the level of transcript accumulation between different cell types. In total, we identified 514 DAS genes; 302 and 276 of these were differently spliced among protonemata and gametophores, and protonemata and protoplasts, respectively (Supplementary Table S2). DAS and DE genes were found to overlap by 4,2% and by 4,6% among protonemata and gametophores, and protonemata and protoplasts, respectively, (Fig. 1C).
There are two types of spliced introns in higher plants: U2-dependent (U2) and U12-dependent (U12) introns52, 53. We calculated that U2-type spliceosomes recognized 99% of AS events (120 181), and U12-type recognized only 1% (1213) in P. patens.
Many eukaryotic genes are alternatively spliced at their 5′-untranscribed region (UTR). In UTRs, AS modulates mRNA stability and thus overall protein expression54, 55. We evaluated the effect of transcripts’ 5′-UTR sequence length on the level of expression of corresponding P. patens gene transcripts. Isoforms with long UTRs were found to have lower expression levels (Supplementary Figure S2).
Changes in Alternative Splicing During Transition from Juvenile to Mature Stages
Three-hundred-and-two genes were found to be DAS between the juvenile life form of the moss and the mature form (Fig. 1C). Functional analysis of these DAS genes revealed that the most frequently represented GO terms were associated with lipid metabolism (GO:0006629), carbohydrate metabolism (GO:0005975), growth and development processes (GO:0080190, lateral growth; GO:0080117, secondary growth), and response to stress conditions (GO:0071329, cellular response to sucrose stimulus; GO:0070417, cellular response to cold) (Fig. 2A; Supplementary Table S2). Examples of DAS genes were Pp1s15_135V6 (ABCG40), Pp1s324_39V6 (poly-(ADP-ribose) polymerase), Pp1s346_35V6 (vacuolar membrane H+-pyrophosphatase), and Pp1s309_69V6 LEA homolog (late embryogenesis abundant domain-containing protein), which are all involved in cellular response to desiccation. These results are in accordance with the specific growth conditions of the gametophore stage. Other DAS genes identified included transcription factors belonging to the AP2/ERF and NAC families (e.g., Pp1s143_82V6 and Pp1s164_37V6), PpABI3A (Pp1s143_82V6), an ABI3 transcription factor homolog, and factors regulating AS such as the SR splicing factor RSP41 (Pp1s144_89V6).
Changes in Alternative Splicing in Response to Protoplastation
Comparing protonemata with protoplasts allowed us to identify 276 DAS genes (Fig. 1C, Supplementary Table S2). Analysis of the GO terms of these genes revealed their involvement in transcription regulation (RNA interference), development (GO:0048441; petal development), cell interactions (GO:0007154; cell communication), and response to biotic (GO:0009615; response to virus) and abiotic (GO:0009628; response to abiotic stimulus) stresses (Fig. 2B).
The DAS genes identified encode proteins with various functions, e.g. a protein kinase superfamily protein (Pp1s22_429V6), translation initiation factor IF2/IF5 (Pp1s626_4V6), transcription initiation factor TFIID subunit 7 (Pp1s111_159V6), regulatory components of ABA receptor 3 (Pp1s97_204V6.2), ARM repeat superfamily protein (Pp1s21_271V6), AP2 ERF domain-containing transcription factor (Pp1s225_102V6), pre-mRNA splicing factor SF2 (Pp1s28_193V6), and SR splicing protein (Pp1s9_310V6).
In protoplasts, we identified 14 genes that had several isoforms with dIF >20 and a considerably increased expression (Supplementary Table S2). For example, Pp1s252_67V6 (A9TKP2) is a putative ATP-binding cassette transporter; its A. thaliana homolog (AT3G28860) is involved in auxin polar transport. We identified four isoforms of this gene; the transcription levels of these isoforms differed considerably between protoplasts and protonemata.
The effect of AS on the Cell Proteome is Small
To estimate the effect of AS on the cell proteome, we predicted 65 846 open reading frames (ORFs) for 58 769 transcripts of 36 233 genes using TransDecoder (https://transdecoder.github.io; see Materials and Methods). We observed that the number of isoforms per gene at transcript level was larger than the number of predicted unique ORFs, thus indicating the greater diversity of mRNAs (Fig. 3A).
We created a custom database of the amino acid sequences corresponding to predicted ORFs and performed mass spectrometry to find proteins encoded by alternatively spliced isoforms. In total, 36 995 unique tryptic peptides from 17 123 predicted ORFs were identified and 7740 translated genes were confirmed. According to our data, 4597 of these genes were alternatively spliced. Overall, we identified 1458 isoform-specific peptides (ISPs) representing 616 (625 ORFs) alternatively spliced genes (Supplementary Table S3). Our approach also identified 13 genes that were not annotated in genome version 1.6. Among these, we identified 85 ISPs from 25 DAS genes (Fig. 3B); however, not all possible isoform-specific proteins could be distinguished from the same locus at the proteome level. This low number of ISP can be explained by following reasons: 1) single isoform of the gene is translated at current time and place; 2) proteins translated from different isoforms of one gene are almost identical, such that the splicing events might result in the addition of extra exons (protein tuning) by alternative transcription initiation (ATI) and alternative transcription termination (ATT) mechanisms, but not in changes to the protein-coding sequences (protein editing). For example, according to our data, the gene encoding gibberellin 3-beta-dioxygenase (Pp1s277_78V6), which is involved in the transformation of non-active gibberellin into the bioactive form, is highly expressed in protoplasts. The Pp1s277_78V6.1 isoform encodes a protein that is 79 amino acids smaller than the Pp1s277_78V6.2 isoform. We identified 16 tryptic peptides, indicating that both isoforms may be translated. However, the unique tryptic peptides, confirming the translation of the Pp1s277_78V6.2 (TCONS_00029818) isoform, were identified only in gametophore and protonema samples (Fig. 3B).
We conducted a search of AS genes for peptides that unambiguously indicated the presence of two or more splice isoforms from the same locus. However, we could only distinguish a few examples, such as Pp1s475_21V6 (amidohydrolase 2) (Supplementary Figure S3; Supplementary Table S3).
In order to exclude poor protein sequence coverage as the main reason for low rates of alternative isoforms detection, we performed in silico AS detection experiment to estimate the number of AS genes we would have expected to identify from our proteomic data (see details in Material and Methods). The test was repeated 100 times and the average number of AS genes from this in silico analysis was 66 times greater than observed in our proteomic experiments (Supplemental Figure S4). This result clearly indicates that the low number of confirmed AS isoforms at proteome level is difficult to explain away as a purely limitation of the proteomics technologies. We also found a low correlation between the level of transcription of alternative isoforms and the number of spectra from isoform-specific peptides (Fig. 3C). Thus, these results showed little correspondence between the predicted proteome plasticity shaped by AS, and observed translation of particular isoforms at the proteome level.
The Possible Role of lncRNAs in the Regulation of Alternative Splicing
Long non-coding RNAs (lncRNAs), which comprise antisense, intergenic, and intron transcripts over 200 nucleotides long, are essential components of eukaryotic transcriptomes56. Interactions of lncRNAs with genomic loci, pre-mRNAs and mRNAs, play an important role in the regulation of gene expression, AS, and translation in mammals. To shed light on the influence of lncRNA on AS we analyzed the potential lncRNA–mRNA and lncRNA–pre-mRNA interactions in the transcriptomes of the three moss cell types.
Firstly, we predicted lncRNAs in gametophore, protonema, and protoplast cells using the strand-specific transcription data obtained previously51 (Fig. 1A). Eight-hundred-and-ninety-seven lncRNAs were found: 679 in gametophores, 592 in protonemata, and 702 in protoplasts (Fig. 4A; Supplementary Table S5). We arbitrarily chose eight identified lncRNAs to validate our RNA-seq data using RT-PCR and qRT-PCR (Supplementary Figure S5, Supplementary Table S4).
From our data set, 897 lncRNAs were used, and 2711 lncRNAs from CANTATAdb50; 145 (15.5%) lncRNAs overlapped the two databases. The analysis revealed 6753 interactions between lncRNAs and mRNA transcripts. Two-hundred-and-ninety-five potential RNA–RNA interactions were found between the 116 lncRNAs from our data set and 159 mRNA transcripts for 109 genes (Supplementary Table S4). The length of the interaction sites ranges from 25 to 4198 bp (Fig. 4B). A highly positive Spearman correlation (r > 0.8, P-value < 0.05) was found between the transcription level of mRNA and the associated lncRNAs for 57 genes suggesting possible co-regulation among them (Fig. 4C, Supplementary Table S4).
We compared the patterns of lncRNA-mRNA interactions for reference transcripts (according to moss genome annotation v1.6) vs. alternative isoforms and found that only 13.5% of interaction sites are common between the two groups of transcripts (Fig. 4D). These data emphasize AS potential to influence RNA-RNA interaction network in the transcriptomes. In addition, our results demonstrated that some of the lncRNA-mRNA interactions are tissue-specific (Fig. 4E).
To evaluate the effect of lncRNAs on AS in moss genes, we analyzed the interactions between lncRNA and pre-mRNA. We detected 5208 potential lncRNA-pre-mRNA interactions between 1221 lncRNAs and pre-mRNA for 1761 genes (Supplementary Table S4). We classified all pre-mRNA sites involved in the establishment of lncRNA-pre-mRNA interactions according to their location on pre-mRNA and found that 14% (720) of interactions occur in potential AS regions. 80% (576) of interactions were identified in the exon–intron junctions implying these sites as the most preferable for lncRNA-pre-mRNA interaction. We also found a significant overrepresentation of alternative splice junctions (Fisher’s exact test, p-value < 2.2e-16) among splice junctions interacted with lncRNAs, suggesting the importance of such interactions in the regulation of AS.
Alterations in Transcription and Splicing of SR proteins
SR proteins regulate alternative splicing in plants and are important for proteome plasticity and gene expression regulation. Moss genome annotation version 1.6 (http://www.cosmoss.org/) revealed 54 gene candidates that are potentially associated with AS processes, 18 of which were SR proteins (Table 1). Changes were analyzed in the transcription and AS of five SR proteins, related to DAS and DE genes in different life forms and treated with plant stress hormones.
According to our data, genes Pp1s9_312V6 and Pp1s28_193V6 (SR34A homolog) differed considerably in their transcription levels (DE genes) between gametophores and protonemata, and Pp1s332_29V6 differed between protonemata and protoplasts. Pp1s9_310V6 (RSZ33) and Pp1s28_193V6 (SR34A) were DAS genes between protonemata and protoplasts, and Pp1s144_89V6 (RS27) was a DAS gene between gametophores and protonemata. RNA-seq data were validated using the RT-PCR and qRT-PCR; the level of AS of these SR genes was estimated using isoform-specific primers in each case (Supplementary Table S5, Supplementary Figure S6).
Detailed analysis of these SR genes by RT-PCR and qRT-PCR is given in Supplementary Data S2 and Supplementary Figure S7. We identified new isoforms of these genes, and data providing evidence of their translation were obtained at the transcriptome and proteome levels.
Transcription and AS of SR genes are affected by stress conditions and hormones16, 21. Gametophores have increased levels of LEA proteins, which are upregulated by ABA, in turn suggesting that there are elevated levels of this hormone in this life form. Previously, we showed that genes participating in the biosynthesis of jasmonic acid are upregulated in protoplasts51. Since this finding suggests that changes in hormonal status are associated with developmental transition or cell reprogramming, we evaluated the influence of ABA and methyl jasmonate (MeJA) on the transcription of specific isoforms of four SR genes using isoform-specific RT-PCR and qRT-PCR (Fig. 4F).
We revealed that treating protonemata with ABA and MeJA leads to changes in the transcription of the specific isoforms of two genes: Pp1s28_193V6 (SR34A homolog) and Pp1s332_29V6 (pp-RSZ23). Pp1s28_193V6 is a DAS gene in protonemata and protoplasts, and a DE gene in protonemata and gametophores. Both isoforms reacted to the treatment of protonemata with ABA in a similar way: the transcription level of each was increased. Interestingly, on treating protonemata with MeJA, the transcription level of the isoform containing a retained intron in the 5′-UTR (TCONS_00033883) increased, while no alteration was seen in the second isoform (TCONS_00030116) with AS in the 5′-UTR (Fig. 4F). This coincides with our classification of this gene as DAS in protonemata and protoplasts, and suggests a possible increase in jasmonic acid derivatives in the latter.
We also analyzed two isoforms of Pp1s332_29V6 (pp-RSZ23), which was upregulated in protoplasts (DE) by isoform-specific primers. The transcription level of both isoforms slightly increased upon treatment with ABA, but there were no changes when treated with MeJA (Fig. 4F). In this gene, no strong influence of ABA or MeJA on AS or transcription was found.
Alternative splicing (AS) is a pivotal mechanism for the post-transcriptional modification of eukaryotic transcripts responsible for transcriptome plasticity and cell proteome diversity57. In this study, we analyzed the transcriptomes of two life forms (gametophores and protonemata) and protoplasts of moss P. patens. We evaluated the AS of genes and identified differentially alternatively spliced (DAS) genes for which the transcriptional level of isoforms of these genes differed considerably between the different cell types. Mass spectrometry was used to evaluate the influence of AS on the cell proteome. We identified 1458 isoform-specific peptides representing 616 alternatively spliced genes. Apparently, our results indicate little correspondence between the predicted proteome plasticity shaped by AS, and the observed translation of particular isoforms at the proteomic level.
Comparing protonemata with gametophores, and protonemata with protoplasts, we found that the splicing of several hundred genes was significantly altered. Our results suggest that AS is regulated at different levels. These findings thus point to the importance of AS in responses to stress conditions and in the development of bryophytes, and seem counter to our findings that AS has no strong influence on the proteome.
The role of alternative splicing in Reprogramming of Protoplast Cells
P. patens protoplasts are a promising model in which to study plant cell reprogramming since they develop into protonemal apical stem cells without needing exogenous hormones47, 58. In our study of protoplasts, the overrepresented GO terms associated with AS genes were those involved in transcription regulation, development, cell communication, and response to stresses. Thus, AS can play important roles in protoplasts reprogramming in moss.
In the transition from the gametophytic to the protoplast stage, expression levels of genes involved in auxin signaling are considerably elevated. For example, the genes involved in auxin polar transport – ABCB4 (Pp1s391_45V6) and ABCB19 (Pp1s252_67V6) – exhibited the highest transcription level on day three of gametophyte development59. According to our data, Pp1s252_67V6 (ppabcb16) is DAS gene; we identified several isoforms with dIF >20. Another DAS gene involved in hormone metabolism is Pp1s277_78V6 (gibberellin 3-beta-dioxygenase), the splicing of which also differed between gametophores and protonemata. In protoplasts, considerable changes were found in the transcription of two isoforms of this gene, which does not seem to correlate with proteome data. It is known that auxins and cytokinins, stimulate protoplasts from different species to reenter the cell cycle, proliferate, and undergo regenerative processes60. We suggest that changes in AS of genes involved in hormone signaling may stimulate protoplast reprogramming and development.
AS influence on cell proteome is overestimated
Eukaryotic genes produce a number of alternative isoforms that are commonly believed to be a major source of cellular protein diversity61,62,63. Although many thousands of alternatively spliced transcripts were detected at the mRNA transcript level, the evidences of AS at the protein level are controversial34, 35, 64, 65. For instance, many of alternative isoforms are subject to cytoplasmic NMD41, 66, 67. Ribosome profiling and mass spectrometry analyses are two main high-throughput instruments to elucidate mRNAs translation into proteins32, 68, 69. Analysis of ribosome profiling data showed that at least 75% of human exon-skipping events were engaged by ribosomes70. However, the recent studies showed that analysis of the same ribosome profiling data might lead to controversial conclusions71, 72. Thus, in spite of the fact that standard mass spectrometry approaches generate relatively low proteome coverage and cannot detect peptides presented at very low levels, large-scale MS experiments are now the main source of evidence of alternative splicing at the protein level. Some of the recent proteomics studies confirmed the considerable amount of AS events at the protein level73, 74. However, it has been shown that these studies dramatically overestimate the number of reliable peptide identifications75. The other proteomic studies in species ranging from human34, 76 to mouse77, rat78, Drosophila79 and Arabidopsis35, 36 confirmed surprisingly small numbers of splice variants. In our study, we performed mass-spectrometry analysis to confirm splicing events in moss cells and could identify only 85 isoform-specific peptides from 25 DAS genes in our dataset. Herewith, we could only distinguish five genes that unambiguously indicated the presence of two or more splice isoforms from the same locus. The expected number of AS genes identified by in silico analysis was substantially larger (66 times) than those in our proteomic experiments. The reasons for low numbers of AS events, detected at the proteome level may be followed: 1) high level of false positives introduced by limitation of accuracy of instruments80, 81; 2) problems with reproducibility of proteomics experiments82; 3) challenges in correct matching of obtained spectra to peptide sequences by search engines83. Post-translational modifications, low-quality spectra or peptide modifications during sample preparation dramatically complicate the peptide identifications84, 85. Taken into account previous studies and our results, we suggest the gap between the number of isoforms detected at mRNA level and the number of proteins confirmed at proteome level is difficult to explain away as purely limitations in proteomics techniques.
On the one hand, AS can lead to the tissue-specific rewiring of protein–protein interaction networks33, on the other hand, stochastic processes may be responsible for creation of alternative isoforms86. We suggest that gene expression levels, not alternative splicing, seem to be the key to proteome diversity in moss and most alternative isoforms have little functional relevance as proteins32. However, there are experimental evidences supporting the tissue-specific rewiring theory and the functional role of AS33, 87, 88. Moreover, it has been shown that N- and C-terminal sequences of protein isoforms are enriched for intrinsically disordered regions (IDRs) and provide regulatory options for tissue-specific and conditional expression89. Apparently, further studies are needed to clarify a functional cellular role of alternative variants.
Regulation of Alternative Splicing by SR Proteins
SR proteins are a family of highly conserved phosphoproteins that play a key role in regulating pre-mRNAs splicing20. In A.thaliana the splicing of SR genes is tissue-specific and is altered in different developmental stages, and by the presence of stress factors and hormones15, 21, 90. However, the effects of various factors on the AS of SR genes in non-vascular plants are unclear. We identified new isoforms for some SR genes, such as Pp1s144_89V6 (RS27), Pp1s9_310V6 (RS2Z33), and Pp1s332_29V6 (pp-RSZ23) (Supplementary Text S2). However, taking into account our proteomic data and known events of unproductive splicing in SR genes, we assume that the majority of these isoforms do not encode intact proteins.
When we compared gametophores and protonemata, our results suggested changes in the AS of genes involved in stress response. It may be assumed that there are specific AS regulators of these genes and, according to our results, one of these factors is the SR gene Pp1s144_89V6.2 (RS27) detected as DAS only when protonemata and gametophores are compared. A homolog of this gene regulates splicing of pre-mRNA and miRNA biogenesis in A. thaliana 91; it is also important in providing a salt-protective response, and its overexpression leads to salt-tolerance13. We suggest that some SR genes regulating stress-response in vascular plants play important roles in moss development.
In protoplasts and protonemata, we identified changes in AS and the transcription levels of Pp1s9_310V6 and Pp1s9_312V6, which are homologous to the SR-protein RSZ33 in Arabidopsis. Overexpression of the gene encoding the RSZ33 splicing factor in A. thaliana leads to phenotypic changes and auxin signaling impairment92. Expression of RSZ33 increases during embryogenesis, and in early stages of sporophyte development92. Predicted by RNA-seq, we identified two new Pp1s9_310V6 isoforms, which differed by an exon at the 5′-UTR, and a new isoform of Pp1s9_312V6. This likely indicates that this protein is important in the regulation of AS during protoplast cell reprogramming.
Upon of the cell with ABA and MeJA, we also found AS changes in Pp1s28_193V6, a homolog of the SR34A protein in A. thaliana. We demonstrated that, in P. patens, isoforms of this gene differ at the 5′-UTR. In A. thaliana, tissue-specific expression of At-SR34a gene isoforms and the repression by exogenous ABA were observed21, 93. Moreover, the At-SR34a gene is regulated by nonsense-mediated decay (NMD), which is in turn linked to splicing events at the 5′-UTR20. These results confirm our RNA-seq data and suggest that ABA in gametophores and MeJA in protoplasts regulate AS and the transcription of SR genes.
AS changes lncRNA-(pre-)mRNAs interaction network
Long non-coding RNAs make up a considerable proportion of the transcriptome of eukaryotic genomes56. It is well known that lncRNAs are enabled of complementary binding to DNA or RNA molecules and direct transcription, post-transcriptional modifications, translation, transport and RNA degradation94. Therefore, the identification of lncRNA binding sites is important for understanding lncRNA based gene regulatory network.
Here we showed that the pre-mRNAs and mRNAs have thousands of potential complementary binding sites for lncRNAs. As the only small fraction of the interaction sites maintains between different isoforms, AS can be considered as one of the major regulator of lncRNA-(pre-)mRNA interaction network. In our study, 14% of the interactions are located in the alternative splicing sites. It was previously shown that antisense intronic lncRNAs may prevent splicing in the corresponding sites95 or change splicing of nearby exons96. Thus, interactions between lncRNAs and pre-mRNA splicing sites may lead to changes in AS. However, the mechanisms regulating AS in trans via lncRNA-mRNA interactions are poorly understood. Interaction sites identified in this study are required for further verification by downstream approaches aimed to silence lncRNAs or change the sequence of interaction sites96. As protocols for genome editing are well optimized for P. patens it can be a very suitable object for further study of functions of lncRNAs in plants.
Material and Methods
Protonema and Gametophore Growth and Protoplast Isolation
Protonemata and gametophores were grown as previously described51. To isolate RNA and protein, 5-d-old protonemata and 8-week-old gametophores were used. Protoplasts were obtained using a modified version of the previously described method51, 97. Protoplast sediment was frozen and used to isolate proteins and RNA.
Sample Treatment with Hormones
Five-d-old protonemata grown on agarized Knop medium were treated with 50 mM ABA or 400 µM MeJA and incubated for 24 h under white light illuminated in a Sanyo Plant Growth Incubator MLR-352H (Panasonic, Osaka, Japan); photon flow was 61 µmol m−2 under a 16-h photoperiod at 24 °C. Protonemata incubated with ultrapure MilliQ water (Merck Millipore, USA) was used as the control. After incubating for 24 h, protonemata were frozen in liquid nitrogen, ground, and used for RNA isolation.
Total RNA Isolation
Total RNA from gametophores, protonemata, and protoplasts was isolated as previously described46. Quality and quantity were evaluated using electrophoresis on agarose gel with ethidium bromide staining. Total RNA concentration of samples was precisely measured using the Quant-iT™ RNA Assay Kit, 5–100 ng on a Qubit 3.0 (Invitrogen, US) fluorometer.
Reverse Transcription and Quantitative Reverse Transcription PCR
cDNA was synthesized using the MMLV RT kit (Evrogen, Russia) according to the manufacturer’s recommendations. Random hexamer primers were used to prepare cDNA from 2 µg total RNA after DNase treatment. Primers were designed using the PrimerQuest Tool (http://eu.idtdna.com/Primerquest/Home/Index) (Supplementary Table S5). For each primer pair, melting curves were analyzed to validate specificity of binding. Real-time PCR was performed using the qPCRmix-HS SYBR system and SYBR Green I (Evrogen) dye on a LightCycler® 96 (Roche, Mannheim, Germany). qPCR was carried out in three biological and three technical replicates. A minus reverse transcriptase (-RT) control contained RNA that had not been treated with reverse transcriptase to confirm the absence of DNA in samples. cDNA representation was normalized using stably transcribed AdePRT (Pp1s212_43V6.1)98, 99.
Transcript Catalogue Assembly
Solid sequencing data was mapped as previously described (Fesenko et al.51). The number of uniquely mapped filtered reads was 31, 36, and 38 million for gametophore, protonema, and protoplast samples, respectively. Mapped RNA-seq data were assembled with Cufflinks v2.2.0100 using Physcomitrella patens genome version 1.6. Cuffmerge was then used to merge assemblies into a master transcriptome, using the reference sequence and reference genome specified above.
Assembly was performed using two versions of Cufflinks, v.2.2.0 and v.2.2.1. Default settings were used, plus –frag-bias-correct, and –multi-read-correct. Seven assemblies were also produced with varying –overhand-toleranсe parameters. Thus, two catalogs were obtained: set1, the Cufflinks v.2.2.0 assembly; and set 2, all transcripts occurring in more than four assemblies, generated using Cufflinks v.2.2.1 with varying –overhand-toleranсe values. Transcription levels were estimated in fragments per kilobase of exon per million fragments mapped (FPKM); only genes with FPKM ≥ 0.2 were used for further analysis. To define the functions of the identified genes, we used moss genome annotation v1.6 (http://www.cosmoss.org) and BLAST search against annotated A. thaliana sequences.
To define the ratio of transcripts spliced by U2 and U12 spliceosomes, we evaluated conserved splice signals in the exon–intron junctions. To do this, we considered the distribution of exons over various splice types in AS genes. Exons flanking the retained introns in the transcripts, and the first exons, were excluded from analysis. This approach identified 121 394 exons, which were further divided into groups. The Biostrings R package was used to evaluate various exon types containing conserved sequences for U2 and U12 spliceosomes. A position-probability matrix (PPM) was used to search for conserved sequences recognized by U2 and U12 spliceosome subunits.
Differential expression was calculated using Cuffdiff software included in the Cufflinks package. Subsequently, differential expression was considered significant if the P-value was greater than the false discovery rate (FDR) after applying the Benjamini–Hochberg correction for multiple-testing (Cuffdiff significance parameter, ‘yes’), and FDR < 0.05. The more stringent rule |log2(FPKMy/FPKMx)| > 2 was used to differentiate genes with high differential expression. To define the functions of the identified genes, we used moss genome annotation 1.6 (http://www.cosmoss.org) and BLAST search against annotated A. thaliana sequences.
spliceR101 was used with default settings to annotate transcripts with classes of AS event. Briefly, based on the information from all transcripts originating from a single gene, spliceR constructs hypothetical pre-RNA and compares it with observed transcripts, thus classifying AS events. For each transcript in each pair (of different life forms or protoplasts), spliceR defined a delta isoform fraction (dIF) value as the difference of the contribution of a transcript to the total gene transcription level.
For all transcription start sites (TSS), Cuffdiff was used to estimate the extent of isoform switching between the isoforms originating from this TSS, as measured by the square root of the Jensen–Shannon divergence computed on the relative abundances of splice variants. Differential splicing was considered significant if P-value and FDR were <0.05 after applying the Benjamini–Hochberg correction for multiple testing.
For all isoforms from significantly differentially spliced TSS, the more stringent rule of absolute value of dIF above 20 was introduced in order to assess the isoforms with the highest levels of differential splicing.
GO Term Analysis
GO groups were adopted from the Physcomitrella patens genome annotation version 1.6. The topGO package in the R environment was used to analyze the enrichment of GO terms in DAS, DEGs, and lncRNA–mRNA groups. Significant GO terms (P-value < 0.05) were selected and results visualized with REVIGO software102.
Identification of Long Non-Coding RNAs
To identify lncRNA candidates, a modified CANTATAdb pipeline was used50. All supposed transcripts from the Cuffmerge assembly with class_code=‘u’, ‘x’, or ‘s’ were selected. Sequences of putative lncRNAs and mRNAs were excised from the genome with samtools, and reverse sequences were reversed and complemented with the revseq –reverse –complement command. The coding potential of the transcripts was evaluated as follows: transcripts over 200 nucleotide long that had not been previously annotated in genome version 1.6 were selected and analyzed for their coding potential using the CNCI program (https://github.com/www-bioinfo-org/CNCI) and Rfam database. Using this approach, transcripts with coding potential (CNCI <0), those with plastid homology (nine transcripts), and mitochondrial genomes (not found) were excluded. To improve the reliability of the data, lncRNAs with FPKM less than 0.5 were discarded in all three cell types. To avoid known RNAs, BLASTN was used on the Rfam plant base; at this stage, the selection criteria were: e-value < 0.00001 and p.ident >97%. Sequences remaining after the filtration stages were used for further analysis as predicted lncRNAs.
Antisense lncRNA may play an important role in the regulation of gene expression. Therefore, we identified cis-natural antisense transcripts (NATs) using the information on overlapping between protein-coding and lncRNAs genes. Eighty-eight lncRNAs were transcribed as cis-NAT, and overlapped with 86 loci of genome version 1.6 (Supplemental Table S4, List 1).
Identification of lncRNA–RNA Interactions
The LAST alignment tool was used since it allows a custom score matrix to be utilized103; this is important because of the presence of so-called ‘wobble pairs’ in addition to canonical compounds. The following values were used in our score matrix: G:C = 4, A:T = 2, G:T = 1; all other cases were termed –6. lncRNAs (query) were aligned on mRNA (database) with the parameters –Q0 –a20 –m100 –s0 –b8, and the –e parameter was evaluated using the lastex tool for each alignment103. For further analysis, lncRNA transcripts with an expression level >0.5 FPKM were used, and mRNAs with an expression level >1 FPKM, in at least one sample.
In silico AS detection experiment
We performed an in silico simulation to estimate the number of AS genes we would expect to detect in the experiments as described previously34. For the simulation, we took genes that were identified in our MS data and assumed that all protein isoforms were expressed equally. We generated tryptic peptides from custom database of predicted ORFs by in silico trypsinolysis. Then we selected at random the same number of peptides for each gene as we identified in our MS data. We calculated the number of genes for which two or more protein isoforms can be unambiguously distinguished by found isoform specific peptides. This procedure was repeated 100 times.
Protein Extraction and Trypsinolysis
Proteins were isolated using the phenol extraction method as previously described104. Four biological repeats for gametophores, four for protonemal and two for protoplast samples were used.
Tryptic peptides were analyzed on a TripleTOF 5600+ (ABSciex, Canada) quadrupole time-of-flight mass spectrometer equipped with a NanoSpray III ion source combined with a NanoLC Ultra 2D+ nano-HPLC (Eksigent) system as previously described51.
Protein database was generated by TransDecoder (http://transdecoder.sourceforge.net/) from the assembled transcripts. TransDecoder was also used for a homology search with the available protein sequences and presence of the known domains using Uniref90 and pfam databases respectively. Raw LC-MS/MS data were converted to peaklists with ProteinPilot 4.5 revision 1656 (Sciex, Canada) using a standard set of identification settings over the protein database. To identify proteins, peaklists were analyzed with MASCOT (version 2.2.07) and X! Tandem (CYCLONE, 2013.2.01) search engines against the protein database with the concatenated reverse decoy dataset. The precursor and fragment mass tolerance were set at 20 ppm and 0.04 Da, respectively. Database-searching parameters included the following: tryptic digestion with one possible missed cleavage, static modifications for carbamidomethyl (C). For X! Tandem we also selected parameters that allowed a quick check for protein N-terminal residue acetylation, peptide N-terminal glutamine ammonia loss, or peptide N-terminal glutamic acid water loss. Result files were submitted to the Scaffold 4 software (version 4.0.7) for validation and meta-analysis. We used the local false discovery rate scoring algorithm with standard experiment-wide protein grouping. For the evaluation of peptide and protein hits, a false discovery rate of 5% was selected for both. False positive identifications were based on reverse database analysis.
Availability of Supporting Data
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE105 partner repository with the dataset identifier PXD005223 and 10.6019/PXD005223.
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This work was supported by the Russian Science Foundation (project No. 14-50-00131).