拟南芥RNA结合蛋白FCA调控转录终止的机制

RNA的表达过程受到多方面因素的调控。来自于酵母和哺乳动物细胞的实验证据表明，在染色质层面即存在复杂的RNA质量控制机制。与动物相比，植物中对染色质水平的转录终止调控以及RNA质量控制的研究还较为初步。在此背景下，本论文主要研究了模式植物拟南芥中一个RNA结合蛋白FCA在调控转录终止及RNA质量控制方面的功能。FCA是植物中最早被鉴定到的RNA结合蛋白之一，也是调控开花的自主途径中的核心蛋白。FCA可以通过抑制FLC (Flowering locus C) 转录，从而促进开花。此外，FCA可以促进其编码基因的提前转录终止从而实现对其自身基因表达的负调控。早期基于基因芯片的数据显示，FCA与RNA结合蛋白FPA同时突变会引起部分基因组内部分位点发生3′ 端转录通读，暗示FCA及FPA可能在转录终止或多聚腺苷酸化位点选择方面发挥重要功能。尽管FCA在调控开花及FLC基因上的调控功能已经基本清楚，但FCA在全转录组水平是否及如何调控转录终止尚待探究。

为了更好地探究FCA如何在染色质水平发挥基因转录相关的调控功能，本研究首先优化建立了一种从植物中分离纯化新生RNA并整合高通量测序的实验方法CB-RNA-seq (Chromatin-bound RNA sequencing)。结果发现，(1) CB-RNA在染色质上的分布沿基因5′ 到3′ 端呈逐渐降低的趋势，且相比成熟mRNA，内含子区域的覆盖度显著升高，说明分离到的CB-RNA是正在转录的新生RNA；(2) 正在进行转录延伸的RNA (Elongating RNAs，RNAe) 的剪接效率显著低于mRNA，说明内含子剪接发生在共转录水平。(3) 已经发生3′ 端转录结束且多聚腺苷酸化的全长RNA (Full-length RNAs，RNAf) 只占CB-RNAs的小部分，其内含子剪接效率低于mRNA，暗示RNAf仍然是新生RNA，需要在染色质上进一步加工将内含子剪接。本研究的结果显示CB-RNA-seq是一种简单高效的研究新生RNA的实验方法。

在建立了CB-RNA-seq方法的基础上，本研究进一步结合了其他多种组学测序及遗传学实验，详细探究了FCA对转录终止及核内RNA质量控制方面的分子机制。(1) 为了从全转录组水平上探究FCA结合RNA的特性，本研究首先通过eCLIP-seq (Enhanced UV crosslinking and immunoprecipitation sequencing) 实验，发现在野生型中FCA特异结合在内含子和pre-mRNA的3′ 端及其下游区域的通读转录本上。(2) 为了探究FCA结合在pre-mRNA 3′ 端的功能，本研究结合pNET-seq实验及Nanopore数据，发现FCA在3′ 端的结合和Pol II的转录终止效率呈负相关，即FCA偏向结合转录终止效率差的pre-mRNA 3′ 端区域；进一步在转录终止缺陷突变体fpa-7的通读转录本上，发现FCA的结合升高，验证了FCA特异结合在转录终止效率低或者异常的转录本上。(3) 为了探究FCA在pre-mRNA 3′ 端区域结合的机制，本研究在RNA 3′ 端加工相关缺失突变体fy-2和fip37-4中，分别发现FCA在3′ 端的结合降低，暗示FCA在pre-mRNA 3′ 端区域的结合部分依赖多聚腺苷酸化信号的识别以及m⁶A (N⁶-methyladenosine) 修饰。(4) 为了探究FCA结合在内含子上的功能，本研究结合CB-RNA-seq实验，发现这些内含子共转录剪接 (Co-transcriptional splicing，CTS) 效率相对较低；当FCA缺失后，这些内含子的剪接效率相对野生型显著降低，说明FCA偏向结合CTS效率较低的内含子，促进共转录剪接。

综上所述，在染色质水平，FCA选择性结合部分共转录剪接效率低的内含子，促进这些内含子的共转录剪接；而在pre-mRNA 3′ 端区域，FCA会偏好结合转录终止效率低的转录本，促进这些转录本的转录终止，而且这种结合部分依赖多聚腺苷酸化信号和m⁶A修饰。本研究对于植物中RNA结合蛋白如何参与转录偶联的RNA加工及RNA质量控制提供了重要理论参考。

RNA expression is regulated by multiple factors. Evidence from yeast and mammalian cells suggests that complex RNA quality control mechanisms exist at the chromatin level. Compared with animals, how transcription termination is regulated and how RNA quality control works at the chromatin level in plants remains largely elusive. This study mainly focused on the function of FCA in transcription termination and RNA quality control in the model plant Arabidopsis. FCA is one of the first RNA-binding proteins identified in plants and a core protein in the autonomous flowering pathway. FCA promotes flowering by inhibiting FLC (Flowering locus C) transcription. In addition, FCA can promote the premature termination of its coding genes, thereby negatively regulating its own gene’s expression. Early microarray-based data showed that simultaneous mutations of FCA and another RNA-binding protein FPA caused transcriptional read-through at the 3′ end of several gene loci, suggesting that FCA and FPA may play important roles in transcription termination or alternative polyadenylation. Although the regulatory function of FCA in flowering has been extensively studied, the questions that whether and how FCA regulates transcription termination on a genome-wide scale remain to be answered.

To explore how FCA works on transcription termination at the chromatin level, this study first optimized the CB-RNA-seq, an experimental method for isolating and purifying nascent RNA from plants and combining it with high-throughput sequencing. The results showed that (1) the reads density of CB-RNA on chromatin declined gradually along 5′ to 3′ of the gene, and compared with mRNA, the coverage of intron region is significantly higher, indicating that the isolated CB-RNAs were nascent RNA; (2) the splicing efficiency of the elongating RNA (RNAe) was significantly lower than that of mRNA, indicating that splicing predominantly occurred at the co-transcriptional level; (3) a small fraction of full-length polyadenylated RNA (RNAf) was included in the isolated CB-RNAs, and its splicing efficiency was lower than that of mRNA, suggesting that RNAf was nascent RNA and further underwent processing steps such as RNA splicing on chromatin. The results of this study show that CB-RNA-seq is a simple and robust experimental method to study nascent RNA.

Taking advantage of the CB-RNA-seq, this study further studied the potential function of FCA in transcription termination and nuclear RNA quality control by applying multi-omics approaches combined with genetics. (1) To explore the features of FCA-bound RNAs at the whole transcriptome level, this study performed FCA-eCLIP-seq. Results showed that FCA bound specifically to the intronic regions and the RNAs correspond to gene 3′ end and downstream of the polyadenylation site. (2) To explore the function of FCA binding at the 3′ end of pre-mRNAs, by combing the pNET-seq experiment and Nanopore data, this study found that the binding of FCA at the 3′ end was negatively correlated with the transcription termination efficiency of Pol II, that was, FCA preferentially bound the 3′ end of pre-mRNAs with poor transcription termination efficiency. Furthermore, in a termination defective mutant fpa-7, FCA binding was significantly enhanced at regions where an increased read-through was observed in fpa-7 compared with that in Col-0. This result verified that FCA binding tended to associate with inefficient or defective termination. (3) To explore the mechanism of FCA binding in the pre-mRNAs 3′ end region, this study found that FCA binding at the 3′ end was reduced in both fy-2 and fip37-4 mutants, suggesting that FCA binding in the pre-mRNA 3′ end region was in part depends on polyadenylation signal recognition and m⁶A (N⁶-methyladenosine) modification. (4) To explore the function of FCA binding at introns, this study found that the co-transcription splicing (CTS) efficiency of FCA-bound introns was relatively low with CB-RNA-seq experiments; when FCA was not functioning, the CTS efficiency of these introns was significantly lower than that of the wild type, indicating that FCA tended to bind introns with low CTS efficiency to promote their CTS.

In summary, FCA selectively binds to introns with low co-transcriptional splicing efficiency promoting co-transcriptional splicing; in the pre-mRNA 3′ region, FCA prefers binding transcripts with low termination efficiency to promote transcription termination, which partially relies on polyadenylation signal and m⁶A modification. This study exemplifies how RNA-binding proteins in plants are involved in transcription-coupled RNA processing and RNA quality control.

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