PTBP1 | Polypyrimidine Tract Binding Protein | 聚嘧啶区结合蛋白 | 关键调控因子

类似之前的EZH2和HDAC1

目的：找PTBP1的target exon（gene）

这个没有生信的办法了，只能根据现有的数据库来找。

Google PTBP1 direct target exon

De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function

待续~

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基本知识回顾：

• 腺嘌呤(A)=胸腺嘧啶(T)，鸟嘌呤(G)=胞嘧啶(C)，所以嘧啶就是指TC，聚嘧啶就是TC聚集在一起的区域。
• Polypyrimidine：The polypyrimidine tract is a region of pre-messenger RNA (mRNA) that promotes the assembly of the spliceosome, the protein complex specialized for carrying out RNA splicing during the process of post-transcriptional modification.
• 脊椎动物（Vertebrate）包括圆口类、鱼类、两栖动物、爬行动物、鸟类和哺乳动物（mammalian）等六大类。出现顺序：鱼-两栖-爬行-鸟-哺乳，都在4亿到1亿年前完成。

PTBP

• 可变剪切的调控因子
• 反式作用因子trans-acting regulators；顺式作用元件，cis-regulatory elements
• heterogeneous nuclear ribonucleoproteins (hnRNPs)
• Binds to the polypyrimidine tract of introns
• Exon 9 encodes an intrinsically disordered and conserved linker between RNA recognition motif 2 (RRM2) and RRM3【就是一个linker而已吗？】
• Sequences overlapping RRM2 and exon 9 in PTBP1 define a minimal splicing repressor domain (12).【剪切抑制域】
• RNA recognition motif (RRM) is a putative RNA-binding domain of about 90 amino acids that are known to bind single-stranded RNAs.
• May promote RNA looping when bound to two separate polypyrimidine tracts in the same pre-mRNA
• Activates exon skipping of its own pre-mRNA
• Plays a role in pre-mRNA splicing and in the regulation of alternative splicing events.
• PTBP1 represses a network of neural-specific alternative exons in non-neural cells and tissues (8, 13, 14).【在非神经组织里，抑制neural exon的AS】
• Activation of this network is required for neuronal differentiation and depends on PTBP1 silencing by miR-124 (8, 13, 14).【miR-124可以通过抑制PTBP1来恢复激活neural exon的AS】
• Many PTBP1-repressed exons are activated by the neuronal-specific Ser/Arg (SR)–related protein of 100 kD (nSR100/SRRM4) (15, 16).【nSR100具有跟PTBP1类似的作用】
• skipping of PTBP1 exon 9 reduces its repressive activity【核心结论，正常tissue里会skip exon9，从而减少抑制活性，才能激活neural pathway】
• PTBP1‐targeting microRNAs regulate cancer‐specific energy metabolism through the modulation of PKM1/M2 splicing【PTBP1与能量代谢的关系】

skipping of polypyrimidine tract–binding protein 1 (PTBP1) exon 9 alters the splicing regulatory activities of PTBP1 and affects the inclusion levels of numerous exons.

During neurogenesis, skipping of exon 9 reduces PTBP1 repressive activity so as to facilitate activation of a brain-specific AS program.

a single exon-skipping event in an RNA binding regulator directs numerous AS changes between species

PTBP1 exon 9 in humans, exon 8 in mice

逻辑：

有没有exon9，PTBP1的活性有显著差别。有exon9时，PTBP1活性【抑制性】增强，从而导致很多其他基因的exon被抑制，最终抑制了神经系统的发育。【有点绕】

理一下：正常情况下，exon9是skipping的，从而导致PTBP1没有抑制性，所以neuronal exon得以激活。

skipping of PTBP1 exon 9 reduces its repressive activity

PTBP1 target exons

待续~

参考文献：

An alternative splicing event amplifies evolutionary differences between vertebrates - Science - 2015

The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation - eLife - 2015 

A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons - Genes Dev - 2007

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读文献，为什么这项研究能发表在Science上，做了些什么工作，有什么核心发现。

An alternative splicing event

evolutionary differences

问题：

• Comparative transcriptomic analyses revealed that vertebrate organ alternative splicing (AS) patterns diverged more rapidly than gene expression differences (4–6). 【评价指标是什么？】
• These AS differences were largely attributed to changes in the use of conserved cis-regulatory elements (4, 5).【不懂，AS跟顺式作用元件有什么关系】
• Cis-regulatory elements are often binding sites for one or more trans-acting factors.
• However, a small number of lineage- and species-dependent AS changes were detected in nucleic acid–binding proteins, suggesting that splicing differences in trans-acting regulators also contributed to the extensive diversity found among vertebrates.【大概有一点懂了】
• The percentage of transcripts with a sequence spliced in [percent spliced in (PSI)] was estimated for cassette exons [including 3 to 27 nucleotide microexons (9)], alternative 5′/3′ splice sites, and more complex AS events.【需要好好看他们是怎么处理的】
• The percentage of transcripts with an intron retained (PIR) was estimated for intron-retention events (10).【PSI和PIR有什么区别？】

做了什么：

• To investigate the role of mammalian-specific exon 9 skipping, we performed RNA sequencing (RNA-seq) to profile AS in 293 cells engineered to express 3xFLAG-tagged PTBP1 transgenes with exon 9 included (PTBP1+Ex9) or deleted (PTBP1-Ex9) (Fig. 1B and fig. S1B).【测了不同处理的cells的RNA-seq，分析了AS。组别：siCtrl, siPTBP1/2, PTBP1+Ex9 and  PTBP1∆Ex9】
• AS events displaying an absolute PSI or PIR of ≥15% upon knockdown of PTBP1 and PTBP2, as well as a “rescue” of ≥50% of the levels observed in the control siRNA treatment, were analyzed further【过滤标准】
• Comparison of the distributions of cassette exon PSI values after induction of each PTBP1 isoform reveals that exon 9 inclusion results in significantly greater restoration of PSI values for both repressed (Fig. 1C, top panel) and stimulated exons (Fig. 1C, bottom panel)【restoration是什么意思？】
• Moreover, titration experiments confirmed that PTBP1+Ex9 and PTBP1DEx9 impart their distinct regulatory activities due to the presence and absence of exon 9 rather than differences in relative expression【调控差异不是基因表达导致的，而是可变剪切】
• Collectively, these results indicate that exon 9 possesses splicing regulatory activity that is partially separable from the repressive activity conferred by RRM2 and that skipping of exon 9 reduces the negative and positive regulatory activities of PTBP1 without substantially affecting RNA binding activity.【一个核心结论，exon 9和RRM2的功能具有相对独立性】

• To investigate whether exon 9 inclusion influences splicing of PTBP1 target exons in other cell types, we investigated whether its PSI correlates significantly with PSI values of isoform dependent and -independent target exons (fig. S7A) across 64 diverse human cell and tissue types (fig. S7B).【开始做cell type的分析，isoform dependent and -independent target exons是什么意思？】
• We observed a significantly higher correlation of exon 9 PSI with PSI of isoform-dependent target exons versus PSI of isoform-independent target exons or exons not regulated by PTBP1 (Fig. 2A, left panel)
• These differences in correlation are not a consequence of isoform-dependent exons having a greater sensitivity to overall PTBP1 levels, because both isoform-dependent and -independent exons display a similar correlation with total PTBP1 mRNA levels (Fig. 2A, right panel)
• Moreover, exon 9 PSI values do not correlate significantly with PTBP1 mRNA levels (fig. S7C)

• Exons with inclusion levels that significantly correlate with the PSI of exon 9 (P < 0.05, Pearson’s product-moment correlation) are enriched in genes functionally associated with cytoskeleton (e.g., FLNB and MYO18A) and nervous system development (e.g., APP and APBB2)

• Thus, mammalian-specific skipping of exon 9 modulates the splicing levels of a large number of functionally coherent PTBP1 target exons across diverse cell and tissue types.【核心结论】

• Given that skipping of PTBP1 exon 9 reduces its repressive activity, we considered that this change might have evolved to further silence PTBP1 and render target exons permissive to activation by nSR100. To investigate this, we employed an in vitro model in which mouse embryonic stem (mES) cells are differentiated into cortical glutamatergic neurons (fig. S8, A and B).【检验skipping of exon9是否会继续降低PTBP1表达，同时激活nSR100】

• Concomitant with reduction of Ptbp1 mRNA levels, skipping of Ptbp1 exon 8 (orthologous to human exon 9) progressively increases during neuronal differentiation of two independent mES cell lines (CGR8 and R1) (Fig. 3, A and B, and fig. S8D).【在神经系统分化过程中，Ptbp1基因表达降低，同时exon 8 skipping增强】

• The transition from skipping to inclusion of exons that are negatively regulated by Ptbp1 and positively regulated by nSR100 begins between neuroepithelial stem cells and radial glia (16).【两个AS调控因子】

• These results suggest that preferential expression of Ptbp1-Ex8 further derepresses the Ptbp1 and nSR100 coregulated neural AS network.【】

• To confirm whether Ptbp1-Ex8 is a weaker repressor than Ptbp1+Ex8, we used in vitro splicing assays to compare the ability of each isoform to compete with nSR100.【】

• To confirm whether Ptbp1 exon 8 skipping affects the kinetics of activation of neural exons in vivo, we used the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system to create mES cell lines that constitutively and uniquely express Ptbp1+Ex8 or Ptbp1-Ex8 (Fig. 3E and fig. S9A).

• Skipping of Ptbp1 exon 8 is therefore important for the activation of target exons during neuronal differentiation.

• Having established that skipping of exon 9 (or exon 8) reduces the regulatory activity of PTBP1 (or Ptbp1) in mammalian cells, we investigated whether introducing this mammalian-specific AS event in a nonmammalian context is sufficient to alter regulation of PTBP1 target exons.【跳过exon9会降低PTBP1的活性，接下来要探究在非哺乳类里面的反应】

• These results support the conclusion that an evolutionary change in PTBP1 exon 9 (or exon 8) inclusion is sufficient to alter the regulation of many exons, including those with important developmental functions.