Title:
Position-specific intron retention is mediated by the histone methyltransferase SDG725
位置特异性内含子保留由组蛋白甲基转移酶SDG725介导
Abstract:
Background: Intron retention (IR), the most prevalent alternative splicing form in plants, plays a critical role in gene expression during plant development and stress response. However, the molecular mechanisms underlying IR regulation remain largely unknown.
背景：内含子保留（IR）是植物中最普遍的选择性剪接形式，其在植物发育和应激反应的基因表达中起着关键作用。 然而，IR调控的分子机制在很大程度上仍然未知。
Results: Knockdown of SDG725, a histone H3 lysine 36 (H3K36)-specific methyltransferase in rice, leads to alterations of IR in more than 4700 genes. Surprisingly, IR events are globally increased at the 5′ region but decreased at the 3′ region of thegenebodyinthe SDG725-knockdown mutant. Chromatin immunoprecipitation sequencing analyses reveal that SDG725 depletion results in a genome-wide increase of the H3K36 mono-methylation (H3K36me1) but, unexpectedly, promoter-proximal shifts of H3K36 di- and tri-methylation (H3K36me2 and H3K36me3). Consistent with the results in animals, the levels of H3K36me1/me2/me3 in rice positively correlate with gene expression levels, whereas shifts of H3K36me2/me3 coincide with position-specific alterations of IR. We find that either H3K36me2 or H3K36me3 alone contributes to the positional change of IR caused by SDG725 knockdown, although IR shift is more significant when both H3K36me2 and H3K36me3 modifications are simultaneously shifted.
结果：在水稻中敲除组蛋白H3赖氨酸36（H3K36）特异性甲基转移酶SDG725会致使超过4700个基因的IR改变。 令人惊讶的是，IR事件会在5’区域全局增加，但在SDG725敲除的突变体基因的3’区域减少。 染色质免疫沉淀测序分析显示，SDG725敲除将导致H3K36单甲基化（H3K36me1）的全基因组增加，但出乎意料地，其也会导致H3K36二甲基化和三甲基化（H3K36me2和H3K36me3）的启动子近端移位。 与动物中的结果一致，水稻中H3K36me1 / me2 / me3的水平与基因的表达水平正相关，而H3K36me2 / me3的位移与IR的位置特异性改变一致。 我们发现单独的H3K36me2或H3K36me3对SDG725敲除引起的IR位置变化有贡献，但当H3K36me2和H3K36me3修饰被同时移除时，IR移位更为显著。
Conclusions: Our results revealed that SDG725 modulates IR in a position-specific manner, indicating that H3K36 methylation plays a role in RNA splicing, probably by marking the retained introns in plants.
结论：我们的结果显示SDG725以位置特异性的方式调控IR，表明H3K36甲基化可能通过标记植物中保留的内含子在RNA剪接中起作用，。
Background:
Intron retention (IR), a specific form of pre-messenger RNA (pre-mRNA) alternative splicing (AS), has attracted increasing attention given its role in global gene expression regulation in both animals and plants [1–6]. Notably, IR is the most prevalent form of AS in higher plants, possibly due to the shorter intron length in plants than in animals [7, 8]. Genome-wide analyses revealed that greater than half of the AS events in rice belong to IR [9, 10].
内含子保留（IR）是一种特定形式的前体信使RNA（前mRNA）选择性剪接（AS），因其在动物和植物中对全局基因表达调控的作用而引起越来越多的关注。 值得注意的是，IR是高等植物中最常见的AS形式，这可能是由于植物内含子长度短于动物所造成的。 全基因组分析显示，水稻中有超过一半的AS事件属于IR。
Several studies have highlighted the functional importance of IR in plants. For example, IR has been shown to associate with abiotic stress response in barley [5, 11]. In strawberry, the level of IR is significantly reduced in post-fertilization compared to prefertilization, suggesting the involvement of IR in fruit maturation [12]. Retention of an intron in the 5′ UTR of the Zinc-Induced Facilitator 2 gene (ZIF2) enhances zinc tolerance in Arabidopsis [6]. Two intron-retained transcripts in Arabidopsis have been shown to remain in the nucleus to avoid nonsense-mediated degradation (NMD)[13]. During Arabidopsis gametophyte development, IR regulates translation in a transcription-independent and spliceosome-dependent manner [14]. Taken together, all these findings underscore the importance of IR in plant growth and development.
一些研究强调了IR在植物中的功能重要性。 例如，已经证明IR与大麦中的非生物胁迫反应有关。 在草莓中，与受精前相比，受精后的IR水平显著降低，表明IR参与了果实成熟。 在ZIF2的5’UTR中，内含子保留增强了拟南芥中的锌耐受性。 已经显示拟南芥中的两个内含子保留的转录本可以留在细胞核中以避免无义介导的降解（NMD）。 在拟南芥配子体发育期间，IR以转录独立性和剪接依赖性方式调控翻译过程。 综上所述，所有这些发现都强调了IR在植物生长和发育中的重要性。
Epigenetic regulators are known to be involved in AS regulation [4, 15, 16]. Histone modifications are particu-larly interesting because of their potential links between chromatin structure and co-transcriptional pre-mRNA splicing. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses in human and mouse cells showed that H3K36me3 (tri-methylation of histone H3 at lysine 36) signals in introns and alternatively spliced exons are considerably lower than those in constitutive exons, suggesting that H3K36me3 modification likely acts as a mark for exons in animal cells [17]. Furthermore, H3K36me3 participates in AS by recruiting the splicing factor polypyrimidine tract-binding protein (PTB) via MRG15, an H3K36me3 reader protein in human [16]. BS69, a specific reader protein for H3.3K36me3, is involved in pre-mRNA splicing, especially IR, by inter-action with the U5 small cytoplasmic fractionation extraction ribonucleoprotein (snRNP) in human cells [4]. Pajoro et al. discovered that H3K36me3 played a role in AS and flowering control in Arabidopsis, wherein mutants of SDG8 and SGD26, two methyltransferases of H3K36, affect temperature-dependent flowering [18]. All these findings support the notion that H3K36me3 plays a direct role in regulating AS.
已知表观遗传调控因子可以参与AS调控。组蛋白修饰因其在染色质结构和共转录前mRNA剪接之间存在潜在的联系，而引起广泛兴趣。人和小鼠细胞中的染色质免疫沉淀测序（ChIP-seq）分析显示，内含子和选择性剪接外显子中的H3K36me3信号要显著低于组成型外显子中的信号，表明H3K36me3修饰可能是动物细胞中外显子的标记。此外，H3K36me3通过MRG15（人体中的H3K36me3读码蛋白）募集剪接因子聚嘧啶束结合蛋白（PTB）来参与AS。 BS69是H3.3K36me3的特异性读码蛋白，其通过与人类细胞中U5小细胞质分级提取核糖核蛋白（snRNP）相互作用，参与mRNA前体的剪接，尤其是IR。 Pajoro等人发现，H3K36me3在拟南芥的AS和开花调控中起作用，其中SDG8和SGD26（H3K36的两个甲基转移酶）的突变体，可以影响温度依赖性的开花。所有这些发现都支持H3K36me3在调控AS中起直接作用的观点。
However, it remains unclear whether all three forms of H3K36 methylation (mono-, di-, and tri-) are involved in AS regulation, especially IR, in plants. We previously reported that two H3K36-specific methyltransferases, SDG725 and SDG708, modulate gene transcription and affect rice growth and development [19–21]. In this study, we investigated splicing alterations in the SDG725- and SDG708-knockdown rice mutants by RNA sequencing (RNA-seq). We found that knockdown of SDG725 led to altered IR in thousands of genes. In addition, IR events tend to increase at the 5′ portion but decrease at the 3′ part of the gene body when comparing the SDG725-knockdown mutant to the wild-type (WT) plants. The results coincided with a higher H3K36me2 occupancy at the 5′ part but a lower one at the 3′ part of the gene body. In contrast, SDG708 knockdown did not cause either these promoter-proximal shifts of IR or histone modification. Our work discovered a previously unknown shift of IR and its possible epigenetic regulator in rice.
然而，我们目前尚不清楚所有三种形式的H3K36甲是否均参与到植物中的AS调控中，尤其是IR。我们以前报道过两种H3K36特异性甲基转移酶SDG725和SDG708可以调节基因转录并影响水稻的生长和发育。在本研究中，我们通过RNA-seq研究了SDG725和SDG708敲除后的水稻突变体中的剪接变化。我们发现SDG725的敲除会导致数千个基因的IR发生改变。此外，当将SDG725敲除突变体与野生型（WT）植物进行比较时，IR事件倾向于在gene body的5’端增加而在gene body的3’端减少。这一结果与gene body5’端较多的H3K36me2、3’端较少的H3K36me2占据率相吻合。相比之下，SDG708的敲除不会引起IR或组蛋白修饰的启动子的近端移位。我们的工作发现了以前未知的IR转换及其在水稻中可能的表观遗传调控因子。
Results:
SDG725 regulates a global shift of intron retention in rice
SDG725调控了水稻内含子保留的全局转变
Since H3K36 methylation has been proposed for splicing regulation in animals, we sought to investigate whether a similar mechanism is also employed in plants. We took advantage of two transgenic rice lines previously gener-ated, in which SDG725 or SDG708 was efficiently knocked down by RNA interference [19, 21]. Quantitative mass spectrometry showed that knockdown of SDG725 led to an increased level of H3K36me1 modification, but decreased levels of both H3K36me2 and H3K36me3 (Additional file 1: Figure S1) [12]. Two biological replicates of RNA-seq libraries were constructed, sequenced, and analyzed for 725Ri-1 (a stable RNAi line of SDG725), 708Ri-1 (a stable RNAi line of SDG708), and WT rice plants (Additional file 2: Table S1) [19, 21]. As the result of SDG725 knockdown, RNA-seq analyses revealed that 462 and 496 genes were up- and down-regulated, respect-ively (Additional file 1: Figure S2a). Gene ontology analysis showed that the differentially expressed genes (DEGs) in 725Ri-1 are enriched in metabolic and biosynthetic pro-cesses (Additional file 1: Figure S2b). The DEGs in 708Ri-1 (245 up- and 222 down-regulated) also are enriched in metabolic processes, but only a small fraction of them overlapped with those found in 725Ri-1 mutant plants [21], indicating the distinct biological roles of these two H3K36-specific methyltransferases in rice.
之前我们已经提出H3K36甲基化可参与动物的剪接调控，所以在本研究中，我们试图研究在植物中是否也有类似的调控机制。我们利用了先前产生的两个转基因水稻品系SDG725和SDG708，其均被RNA干扰有效地敲除。定量质谱显示，SDG725的敲除会导致H3K36me1修饰的增加，但会导致H3K36me2和H3K36me3修饰的降低（附加文件1：图S1）。我们对725Ri-1、708Ri-1和WT水稻野生型的各两个生物学重复进行了RNA-seq建库、测序和分析。作为SDG725敲除的结果，RNA-seq分析显示，分别有462和496个基因上调和下调（附加文件1：图S2a）。GO分析显示，725Ri-1中的差异表达基因（DEG）主要富集在代谢和生物合成过程中（附加文件1：图S2b）。 708Ri-1中的DEGs（245上调和下调222下调）也富集在代谢过程，但只有一小部分与725Ri-1突变体中的DEG相重叠，暗示出这两种H3K36特异性甲基转移酶在水稻中的独特的生物学作用。
The splicing changes in rice were further analyzed by the SplAdder approach [22]. Consistent with previous findings [23], IR is the predominant form of AS events identified by comparing to rice genome annotation (Additional file 2: Table S2). To perform a quantitative investigation on IR, we used the intron retention index (IRI) to estimate the extent of retention for each annotated in-tron [3]. We found that 4714 genes contain one or more altered IR, including 2089 IRI-up introns (≥ twofold in-crease in IRI) and 4214 IRI-down introns (≥ twofold de-crease in IRI) by comparing the 725Ri-1 plants to the WT rice plants. Ten IRI-altered introns were selected for validation by reverse transcription followed by quantitative polymerase chain reaction (qRT-PCR). Primer pairs were designed to detect either spliced or intron-retained transcripts for each IR event (Additional file 2: Table S3). For all 10 cases, qRT-PCR results confirmed the RNA-seq data (Fig. 1a). To our surprise, we found that the IRI-up introns were favored at the 5′ portion of the gene body while the IRI-down introns were preferred at the 3′ portion of the gene body when comparing 725Ri-1 to WT rice plants using stringent coverage requirements (Fig. 1b, Additional file 1: Figure S3). The conclusion remained the same when the requirements of intron length coverage were relaxed (Additional file 1: Figure S4). In contrast, no location bias was observed between the 3326 IRI-up and 2497 IRI-down introns in 708Ri-1 (Fig. 1c, Additional file 2:Table S4). For exon skipping, no obvious position-specific splicing alteration was found by comparing 725Ri-1 with WT rice. Our results showed that knockdown of SDG725 but not SDG708 alters IR in a position-specific manner.
我们通过SplAdder方法进一步分析了水稻中的剪接变化。与先前的水稻比较基因组学的注释结果一致，IR是水稻中AS事件的主要形式（附加文件2：表S2）。为了对IR进行定量研究，我们使用内含子保留指数（IRI）来估计每个经注释的intron的保留程度。通过比较725Ri-1与WT，我们发现有4714个基因含有一个或多个改变的IR，包括2089个IRI-up内含子（在IRI中≥2倍增加）和4214个IRI-down内含子（在IRI中≥2倍降低）。我们选择了10个IRI改变的内含子，并通过逆转录然后对其进行了qRT-PCR的验证。我们设计引物对，用于检测每个IR事件的剪接或内含子保留的转录本（附加文件2：表S3）。对于所有10个基因，qRT-PCR的结果均证实了RNA-seq数据的结果（图1a）。令人惊讶的是，当我们使用严格的覆盖要求，将725Ri-1与WT比较时发现，IRI-up内含子在gene body的5’端受到偏好，而IRI-down内含子在gene body的3’端受到偏好（图1b，附加文件1：图S3）。当放宽内含子长度覆盖的要求时，此结论仍然保持不变（附加文件1：图S4）。相反的是，我们没有在708Ri-1中的3326个IRI-up和2497个IRI-down内含子之间观察到位置偏差（图1c，附加文件2：表S4）。对于外显子跳跃，我们通过比较725Ri-1和WT水稻，但是并没有发现明显的位置特异性的改变。我们的结果显示，SDG72的敲除（而非SDG708）会以位置特异性的方式改变IR。
Genes with increased IR show reduced expression levels
IR增加的基因表达水平降低
Among all the genes with one or more retained introns, 1399 genes only contain IRI-up introns, 2908 genes only contain IRI-down introns, and 407 genes contain both IRI-up and IRI-down introns (Fig. 2a). IR may regulate gene expression through several different mechanisms, including but not limited to the following: (1) intron-retained transcripts are unstable, being degraded by nuclear RNA surveillance machinery or cytoplasmic NMD [3, 13, 24–26]; (2) intron-retained transcripts are stable and can be translated into new protein variants, where those introns are known as “exitrons” [27–29]; (3) a retained intron in a 5′ UTR regulates translation initi-ation [30] while IR in a 3′ UTR can either repress mRNA stability and translation by introducing more regulatory cis elements [31] or stabilize mRNA by avoiding NMD [32]. Therefore, global IR coupled with RNA stability could serve as a novel mechanism to finetune gene expression in rice. RNA-seq data were then used to determine the effect of altered IR on gene expression. We found that the expression level of the genes with only IRI-down introns tends to be up-regulated compared to those with only IRI-up introns, and the expression changes of the 407 genes with both IRI-up and IRI-down introns fall in between (Fig. 2b). These observations agreed with the notion that IR may serve as a post-transcriptional mechanism to reduce gene expression [1–4]. Consistent with Fig. 1b,inthe 407genes,IRI-up introns show a similar preference at the 5′ part of the gene body and IRI-down introns are enriched at the 3′ end of the gene body (Fig. 2c). The same occurs with the genes with IRI-up only and IRI-down only introns (Fig. 2d).
在具有一个或多个保留内含子的全体基因中，1399个基因仅含有IRI-up内含子，2908个基因仅含有IRI-down内含子，407个基因同时含有IRI-up和IRI-down内含子（图2a）。 IR可通过以下几种不同的机制调控基因的表达：（1）内含子保留的转录本不稳定，被核RNA监测机制或细胞质NMD降解; （2）内含子保留的转录本是稳定的并且可以翻译成新的蛋白质变体，其中那些内含子被称为“exitrons”; （3）5’UTR中保留的内含子调控翻译起始，而3’UTR中的IR可通过引入更多的调控顺式元件来抑制mRNA稳定性和翻译或通过避免NMD来稳定mRNA。因此，全局IR与RNA稳定性相结合可以作为微调水稻基因表达的新机制。接下来，我们使用RNA-seq数据来确定改变的IR对基因表达的影响。我们发现，与仅有IRI-up内含子的基因相比，只有IRI-down内含子的基因的表达水平趋于上调，而具有IRI-up和IRI-down内含子的407基因的表达变化的下降介于两者之间（图2b）。这些观察结果与IR可能作为减少基因表达的转录后调控机制的观点一致。与图1b一致，在407基因中，IRI-up 内含子在gene body的5’端显示出相似的偏好，并且IRI-down内含子在gene body的3’端富集（图2c）。我们在仅具有IRI-up内含子和IRI-down内含子的基因中也观察到相同的现象（图2d）。
Intron retention shift correlates with distribution shifts of H3K36 methylations
内含子保留位移与H3K36甲基化的分布变化相关
We next asked why IR is shifted as the result of SDG725 knockdown. Considering the role of H3K36 methylation in IR in animals, we acquired H3K36 mono-, di-, and tri-methylation (H3K36me1/me2/me3) profiles of the 725Ri-1, 708Ri-1, and WT rice plants using ChIP-seq (Additional file 2: Table S5). By looking at gene-dense regions we found that H3K36me2 and H3K36me3 profiles exhibit apparent promoter-proximal shifts in 725Ri-1 but not in 708Ri-1 compared to those in WT rice plants (Fig. 3a). These observations were further con-firmed by genome-wide analyses (Fig. 3b–d). It is worth noting that H3K36me1 levels are increased in nearly the entire gene body (Fig. 3b, left panel) upon SDG725 knockdown. In contrast, SDG708 knockdown led to a global decrease of all three H3K36 methylation marks (Fig. 3a, b).
我们接下来想知道为什么IR会因为SDG725的敲除而移位。 考虑到H3K36甲基化在动物IR中的作用，我们使用ChIP-seq获得了725Ri-1,708Ri-1和WT水稻植物的H3K36单，二和三甲基化谱。 （附加文件2：表S5）。 通过观察基因密集区域，我们发现，与WT水稻植物相比，H3K36me2和H3K36me3表达谱件在725Ri-1中表现出明显的启动子近端移位，但在708Ri-1中却没有这种情况（图3a）。 全基因组分析进一步证实了这些观察结果（图3b-d）。 值得注意的是，在SDG725敲除后，在几乎整个gene body中的H3K36me1水平都有所增加（图3b，左图）。 相比之下，SDG708敲除则会导致所有三种H3K36甲基化标记的全面减少（图3a，b）。
To obtain a detailed view of H3K36 methylation changes at the individual gene level, we computed the dif-ference of ChIP-seq tag density gene by gene between knockdown and WT plants (see details in Methods, Additional file 1: Figure S5). For almost all transcribed loci in the 725Ri-1 plants, H3K36me1 levels are increased across the gene body (Fig. 3c, left panel), while H3K36me2 (Fig. 3c, middle panel) and H3K36me3 (Fig. 3c,right panel) levels are increased at the 5′ end but decreased at the 3′ end of the gene body. However, this histone methylation positional bias was not detected in the 708Ri-1 plants (Fig. 3d), suggesting that the promoter-proximal shifts of H3K36me2/me3 are specific for the 725Ri-1 plants.
为了获得单个基因水平的H3K36甲基化变化的详细信息，我们计算了knockdown和WT植物之间基因对ChIP-seq标签密度基因的差异（图S5中的细节）。 对于725Ri-1植物中几乎所有转录的基因座，H3K36me1水平在gene body中增加（图3c，左图），而H3K36me2（图3c，中图）和H3K36me3（图3c，右图）水平 在5’末端增加，但在gene body的3’末端减少。 然而，我们并未在708Ri-1植物中检测到这种组蛋白甲基化位置偏差（图3d），这表明H3K36me2 / me3的启动子近端移位对725Ri-1植物是特异性的。
H3K36me2/me3 shifts positively correlate with IR shifts caused by SDG725 knockdown
H3K36me2/me3与SDG725敲除引起的IR变化呈正相关
SDG725 knockdown caused global shifts of H3K36me2 and H3K36me3, providing us with a unique opportunity to examine the potential contributor for IR shift. We therefore divided the transcribed genes into four groups. For type I genes, only H3K36me2 show a promoter-proximal shift. Type II genes are designated for H3K36me3 shift towards the 5′ end, while type III genes exhibit pattern shifts for both H3K36 methyl marks. Type IV genes show no shift for either H3K36me2 or H3K36me3. We noticed that H3K36me2 shift alone led to a considerable effect on the location bias of IR events (Fig. 4a). Similarly, H3K36me3 shift alone has an impact on IR shift (Fig. 4b). Intriguingly, when both H3K36me2 and H3K36me3 profiles are simultaneously shifted, IR showed a more significant shift compared to either H3K36me2 or H3K36me3 alone (Fig. 4c). As a control, unshifted H3K36me2/me3 exhibit no significant effect on IR switch (Fig. 4d). These results suggested that both H3K36me2 and H3K36me3 are involved in modulating IR shift, although a collaborative mechanism may exist in regulating IR.
SDG725敲除会导致H3K36me2和H3K36me3的全局移位，这为我们探寻IR移位的潜在因素提供了机会。因此，我们将转录的基因分成四组。I型是仅H3K36me2显示启动子近端移位的基因。 II型为H3K36me3向5’端移位的基因，而III型是表现出两种H3K36甲基标记的移位的基因。 IV型是H3K36me2或H3K36me3没有移位的基因。我们注意到H3K36me2移位会对IR事件的位置偏差产生很大影响（图4a）。类似地，单独的H3K36me3移位也会对IR移位有影响（图4b）。有趣的是，当H3K36me2和H3K36me3的表达谱同时被移位时，与单独的H3K36me2或H3K36me3相比，IR显示出更显著的移位（图4c）。作为对照，未移位的H3K36me2 / me3对IR改变没有显著影响（图4d）。这些结果表明，虽然可能存在一些协作的机制来调节IR，但H3K36me2和H3K36me3都参与了IR移位的调控。
However, the signals of H3K36 mono-, di-, and tri-methylation were also compared in differentially retained introns. Interestingly, both the levels of H3K36me2 and H3K36me3 at IRI-up introns were higher than those at IRI-down introns (Additional file 1: Figure S6), support-ing the notion that H3K36me2/me3 probably demarcate IR in rice.
然而，我们还在差异保留的内含子中比较了H3K36单，双和三甲基化的信号。 有趣的是，IRI-up内含子的H3K36me2和H3K36me3水平均高于IRI-down内含子（附加文件1：图S6），这支持了H3K36me2/me3可能在水稻中调控IR的观点。
Validation of the association between intron retention and H3K36me2 modification
验证内含子保留和H3K36me2修饰之间的关联
The candidate gene approach was used to validate the potential link between H3K36me2 and IR. Two genes (LOC_Os01g24680, encodes putative 3-hydroxyacyl-CoA dehydrogenase; LOC_Os08g01760, encodes putative nu-trition dehydrogenase) were selected from the 407 genes containing both IRI-up and IRI-down introns. For each gene, we selected six regions along the gene body to test H3K36me2 occupancy by ChIP-PCR. In addition, two IR events (one IRI-up and one IRI-down) for both genes were also examined by qRT-PCR in the same samples by multiple primer pairs spanning both intronic location and donor/acceptor sites (Fig. 5a, b, Additional file 2: Table S6). The results confirmed that the chromatin re-gions with increased levels of H3K36me2 modification produce increased IR events; accordingly, the chromatin regions with reduced H3K36me2 occupancy are associated with decreased IR events (Fig. 5).
候选基因方法用于验证H3K36me2和IR之间的潜在联系。 我们从含有IRI-up和IRI-down内含子的407个基因中选出了两个基因（LOC_Os01g24680，编码推定的3-羟基酰基-CoA脱氢酶; LOC_Os08g01760，编码推定的营养酶脱氢酶）。 对于每个基因，我们选择顺着gene body的六个区域，通过ChIP-PCR测试其H3K36me2占据情况。 此外，两个基因的两个IR事件（一个IRI-up和一个IRI-down）也通过qRT-PCR在同一样品中通过跨越内含子位置和供体/受体位点的多个引物对进行检测（图5a，b）。 结果证实，随着H3K36me2修饰水平的增加，染色质区域产生的IR事件也会增加; 类似地，H3K36me2占有率降低的染色质区域与IR事件的减少相关（图5）。
Transcripts with changed IR mainly accumulate in the nucleus
具有改变的IR的转录物主要累积在细胞核中
Cellular fractionation was performed to examine whether the IR transcripts are accumulated in the nu-cleus or cytoplasm. The success of cellular fractionation was confirmed by qRT-PCR of genes specifically expressed in the nucleus or cytoplasm (Additional file 1: Figure S7) [33, 34]. RNA-seq analysis on each fraction showed that transcripts with changed IR in rice domin-antly accumulated in the nucleus (Fig. 6a, b, Additional file 2: Table S7). To further examine whether cytoplas-mic NMD contributes to the transcript abundance, both 725Ri-1 and WT plants are treated with cycloheximide (CHX) to stabilize transcripts that are otherwise degraded by NMD, followed by RNA-seq analysis.
我们对细胞进行了分块，以检查IR转录本是否会在细胞核或细胞质中累积。 通过在细胞核或细胞质中特异性表达的基因的qRT-PCR，我们证实了细胞分块的结果（附加文件1：图S7）。 对每个细胞部分的RNA-seq分析显示出，水稻中具有改变的IR的转录本主要在细胞核中积累（图6a，b，附加文件2：表S7）。 为了进一步检查细胞质NMD是否有助于转录本丰度，我们使用放线菌酮（CHX）处理725Ri-1和WT植物，以稳定转录产物，以防被NMD降解，然后进行RNA-seq分析。
Transcripts with a premature termination codon (PTC) showed an increased steady-state expression level upon CHX treatment (Additional file 1: Figure S8), indicating the successful inhibition of NMD. In contrast, genes with IR did not show an obvious expression increase upon CHX treatment (Additional file 1: Figure S9), suggesting cytoplasmic NMD has limited contribution to overall expression of IR loci in rice. These results indi-cate that intron-retained transcripts in rice are mainly accumulated in the nucleus rather than the cytoplasm and contribute to steady-state gene expression.
具有提前终止密码子（PTC）的转录本在CHX处理后显示出稳态表达水平的增加（附加文件1：图S8），这表明我们成功抑制了NMD。 相反，具有IR的基因在CHX处理后没有显示出明显的表达量的增加（附加文件1：图S9），这表明细胞质NMD对水稻中IR基因座的总体表达的贡献有限。 这些结果表明，水稻内含子保留的转录本主要积聚在细胞核而不是细胞质中，这有助于稳态基因的表达。
Discussion:
IR is thought to play a critical role in gene expression regulation in animals and plants. However, how IR is regulated in plants remains largely unknown. In this study we revealed that SDG725 may regulate global IR through H3K36me2/me3 modifications. We previously reported that SDG725 acts as an H3K36 methyltransferase and functions in promoting gene transcription [19, 20]. In both WT and 725Ri-1 plants, a positive correl-ation was observed between transcript abundance and H3K36me1/me2/me3 levels (Additional file 1: Figure S10a), extending previous findings obtained from other species [17, 35]. We discovered that changes in average H3K36me2/me3 occupancy level positively correlate with expression changes between 725Ri-1 with WT plants (Additional file 1: Figure S10b, c, left panels). The shift of H3K36me2/me3 affects IR but not expression level if the overall H3K36me2/me3 level does not change (Additional file 1: Figure S10b, c, right panels; Additional file 1: Figure S11). Taken together, we propose that SDG725 may control gene expression through two dif-ferent mechanisms: (1) by modulating gene transcription via changing the overall levels of H3K36me2/me3, and (2) by regulating IR shift through H3K36me2/me3 shifts. How H3K36me2 or H3K36me2/me3 modulates IR in rice remains an open question. It might be achieved by reducing Pol II elongation rate and/or through a chro-matin adaptor mechanism. In the first model, increased H3K36me2/me3 levels may slow down the elongation of Pol II. A longer dwell time of Pol II on introns may recruit splicing repressive factors or inhibit positive splicing factors to promote IR [2]. Alternatively, H3K36me2/me3 may be recognized by a specific “reader” protein, which interacts with splicing repressive factors to promote IR [4, 16]. Notably, the two models are not mutually exclusive and may act in concert to recruit splicing regulators. While significant expression change was not detected for known splicing factors be-tween 725Ri-1 and WT rice (Additional file 2: Table S8), further investigations are warranted to identify H3K36me2/H3K36me3 reader(s) as well as downstream factors functionally involved in splicing regulation in rice.
IR被认为在动物和植物的基因表达调控中起关键作用。然而，IR是如何在植物中被调控的，仍然未知。在本研究中，我们发现SDG725可能通过H3K36me2/me3修饰来调控全局IR。我们以前报道过SDG725可以作为H3K36甲基转移酶，并且在促进基因转录中起作用。在WT和725Ri-1植物中，我们观察到转录本丰度和H3K36me1/me2/me3水平之间存在正相关，这扩展了之前在其他物种中的发现。我们发现，H3K36me2/me3占有水平的平均变化与725Ri-1与WT植物之间的表达变化呈正相关。如果整体H3K36me2/me3水平没有改变，那么H3K36me2/me3的移位，虽会影响IR但不影响基因的表达水平。总之，我们提出了SDG725可以通过两种不同的机制控制基因表达的观点：（1）通过改变H3K36me2/me3的总体水平来调节基因转录；（2）通过调节调控H3K36me2/me3移位，致使IR移位。 H3K36me2或H3K36me2/me3是如何在水稻中调控IR的，这仍然是一个亟待解决的问题。它有可能是通过降低Pol II伸长率或通过染色质adaptor机制来实现。在第一个模型中，H3K36me2/me3水平的增加可能会减慢Pol II的延长。对那些停留时间长的内含子，Pol II可能会招募剪接抑制因子或抑制阳性剪接因子来促进IR。另外，H3K36me2/me3可被特异性的“读码”蛋白所识别，其可与剪接抑制因子相互作用以促进IR。值得注意的是，这两种模式并不互斥，其可能会进行互作以招募剪接调控因子。虽然我们没有检测到725Ri-1和WT水稻之间的那些已知的剪接因子在表达方面的显著变化，但我们仍需要进一步研究以鉴定H3K36me2/H3K36me3的读码以及涉及水稻剪接调控的下游因子。
The coupling of transcription and splicing is prevalent [36]. We observed a positive correlation between efficient splicing (or less IR) and gene expression level (Fig. 2b), indicating that IR negatively impacts the steady-state RNA level likely due to a higher degradation of IR transcripts. Moreover, increased IR events in the 5′ half of the gene in the SDG725-knockdown line suggest interaction between transcription and splicing machineries. Besides IR, other factors such as transcrip-tion activity are also possible contributors to steady-state mRNA expression.
转录和剪接的偶联十分普遍。我们观察到有效剪接（或者说是更少的IR）和基因表达水平之间的正相关（图2b），表明IR可能由于其转录本的高降解而对稳态RNA的水平产生负面影响。 此外，SDG725敲除line中，在其基因5’端的一半的IR事件的增加，也表明了转录和剪接机器之间存在相互作用。 除了IR，其他因素也可能是稳态mRNA表达的原因，比如转录活性。
To investigate the characteristics of retained introns, we examined several features including intron length, GC content, and splice site strength. Compared with spliced introns, retained introns tend to have much longer length, higher GC content, and weaker splice strength in the present rice study (Additional file 1: Figures S12–S14) [2, 3, 37]. Notably, the retained introns in animals tend to be shorter compared to spliced in-trons [2]. Because the average intron size is much bigger in animals (9519 bp in mouse and 11,538 bp in human) than in plants (407 bp for rice), we speculate that in animals a shorter intron is more likely to be recognized as an exon and has a higher tendency to be retained, whereas in plants a longer intron has a higher tendency to be recognized as an exon and is subsequently retained. The underlying mechanism is expected to be complicated and deserves further characterization.
为了研究保留内含子的特征，我们检查了以下几个特征，包括内含子长度，GC含量和剪接位点强度。 与剪接的内含子相比，保留的内含子具有更长的长度，更高的GC含量和更弱的剪接强度（附加文件1：图S12-S14）。 值得注意的是，动物中的保留内含子往往比剪接内含子更短。 由于动物的平均内含子（小鼠为9519bp，人类为11,538bp）比植物（大米为407bp）要长得多，所以我们推测在动物中，较短的内含子更可能被认为是外显子。 并且具有更高的保留倾向，而在植物中，较长的内含子极大可能会被识别为外显子，并随后被保留。 潜在的机制应该很复杂，值得进一步的鉴别。
The question of why the SDG708 knockdown shows a global decrease of H3K36me2/me3 while the SDG725 mutant displays a pattern shift is intriguing. One pos-sible explanation is the different enzyme specificity between SDG725 and SDG708. Although both are H3K36 methyltransferases, SDG725 plays a major role in mono-to di- and di- to tri-methylation, while SDG708 func-tions more on me0 to mono-methylation according to our previous studies [19, 38]. As expected, knockdown of SDG708 reduced the level of H3K36me1 around the transcription start site (TSS) and transcription termin-ation site (TTS) (Fig. 3b). The occupancy of H3K36me2 and H3K36me3 was also reduced (Fig. 3b), possibly due to the lack of H3K36me1. In addition, SDG725 but not SDG708 contains a CW domain, which can bind to H3K4 methylation that is enriched at promoter regions [39]. When SDG725 became limiting under the knockdown condition, it was preferentially recruited to the 5′ end of the gene, thereby showing more reductions of di- and tri-methylation of H3K36 at the 3′ portion of the gene body. This explains that the ChIP-seq result, rep-resented as a relative distribution instead of an absolute level, showed a relative decrease of H3K36me2/me3 level in the 3′ half of the gene body but a relative increase of H3K36me2/me3 level close to the 5′ end (Fig. 3b).
当SDG725突变体显示模式转换时，为什么SDG708的敲除会显示出H3K36me2/me3的全局减少呢？这一问题十分有趣。一个可能的解释是SDG725和SDG708之间的不同酶特异性。虽然两者都是H3K36甲基转移酶，但SDG725在单、双和三甲基化中起主要作用，而SDG708在me0单甲基化上的功能更多。正如预期的那样，SDG708的敲除降低了转录起始位点（TSS）和转录终止位点（TTS）周围的H3K36me1水平（图3b）。 H3K36me2和H3K36me3的占有率也有所降低（图3b），这可能是由于缺乏H3K36me1所致。此外，SDG725而非SDG708含有CW结构域，其可以结合富集于启动子区域的H3K4甲基化上[39]。当SDG725在敲除条件下表达量被限制时，它优先被招募到基因的5’末端，进而显示出gene body的3’端H3K36的二甲基化和三甲基化的更多减少。这解释了ChIP-seq结果，代表一种相对分布而非绝对水平，显示出H3K36me2/me3水平在gene body3’端一半区域的的相对降低，以及在gene body5’端一半区域的的相对增加（图3b）。
Several lines of evidence support the notion that H3K36me2 in rice probably serves as the functional counterpart of H3K36me3 in animal cells [4, 16, 17]. H3K36me2 in plants and H3K36me3 in animals share similar occupancy profiles at transcribed regions (Fig. 3)[21]. The H3K36me3 patterns in both rice and Arabidop-sis resemble the H3K4me3 or H3K79me3 profile in ani-mals (Additional file 1:FigureS15)[40–43]. These observations suggest that plants and animals may employ distinct epigenetic factors (e.g., writers and reads) to co-ordinate transcription and post-transcriptional gene regulation, although the underlying regulatory principles are conserved during evolution. Our study demonstrated for the first time that a histone methyltransferase can regulate the position preference of IR,a unique phenomenon that deserves further molecular characterization.
目前有几个证据可以支持水稻中的H3K36me2在功能方面可能与动物细胞中的H3K36me3相对应的观点。 植物中的H3K36me2和动物中的H3K36me3在转录区域具有相似的占据谱（图3）。 水稻和拟南芥中的H3K36me3模式类似于动物中的H3K4me3或H3K79me3谱（附加文件1：图15）。 这些观察结果表明，植物和动物可以采用不同的表观遗传因子来协调转录和转录后基因调控，尽管潜在的调控准则在进化过程中相当保守。 我们的研究首次表明，组蛋白甲基转移酶可以调控IR的位置偏好，值得进行进一步的分子层面的表征。
Conclusions:
We found that depletion of the histone methyltransferase SDG725 in rice leads to position-specific alteration of in-tron retention (IR), a phenomenon that has not been dem-onstrated previously in any species. Further analyses support the model that H3K36me2/me3 but not H3K36me1 contribute to the IR alteration. As IR plays an important role in regulated gene expression of both plants and animals, the position-specific IR regulation revealed by this study further extends our knowledge regarding the complexity of gene regulatory networks.
我们发现水稻中组蛋白甲基转移酶SDG725的敲除导致了位置特异性的内含子保留（IR）的改变，这种现象在以前的任何物种中都没有得到证实。进一步分析支持了H3K36me2/me3（而非H3K36me1）对IR改变有贡献的模型。由于IR在植物和动物的基因表达调控中起重要作用，本研究揭示的位置特异性IR调控进一步扩展了我们对基因调控网络复杂性的认识。
