PTBP1 | Polypyrimidine Tract Binding Protein | 聚嘧啶區結合蛋白 | 關鍵調控因子

類似之前的EZH2和HDAC1，但更復雜一點。

 

一些縮寫：

RNA-binding proteins (RBPs)
RNA-binding domains (RBDs)

• RNA recognition motif (RRM)
• hnRNP K-homology (KH) domains

 

目的：找PTBP1的target exon（gene）

這個沒有生信的辦法了，只能根據現有的數據庫來找。

 

Google 

• PTBP1 direct target exon
• PTBP1 RNA binding site

收集到的文獻：

• De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function
• Roles of PTBP1 in alternative splicing, glycolysis, and oncogensis
• The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation

Douglas L. Black這個大佬做AS很厲害，文章也發得很好，但貌似不喜歡做工具給別人用。

 

待續~

 

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基本知識回顧：

• 腺嘌呤(A)=胸腺嘧啶(T)，鳥嘌呤(G)=胞嘧啶(C)，所以嘧啶就是指TC，聚嘧啶就是TC聚集在一起的區域。
• 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.
• 脊椎動物（Vertebrate）包括圓口類、魚類、兩棲動物、爬行動物、鳥類和哺乳動物（mammalian）等六大類。出現順序：魚-兩棲-爬行-鳥-哺乳，都在4億到1億年前完成。

 

PTBP

• 可變剪切的調控因子
• 反式作用因子trans-acting regulators；順式作用元件，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【就是一個linker而已嗎？】
• 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).【在非神經組織里，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可以通過抑制PTBP1來恢復激活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類似的作用】
• skipping of PTBP1 exon 9 reduces its repressive activity【核心結論，正常tissue里會skip exon9，從而減少抑制活性，才能激活neural pathway】
• PTBP1‐targeting microRNAs regulate cancer‐specific energy metabolism through the modulation of PKM1/M2 splicing【PTBP1與能量代謝的關系】

 

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

 

邏輯：

有沒有exon9，PTBP1的活性有顯著差別。有exon9時，PTBP1活性【抑制性】增強，從而導致很多其他基因的exon被抑制，最終抑制了神經系統的發育。【有點繞】

理一下：正常情況下，exon9是skipping的，從而導致PTBP1沒有抑制性，所以neuronal exon得以激活。

skipping of PTBP1 exon 9 reduces its repressive activity

 

PTBP1 target exons

 

待續~

 

 

參考文獻：

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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讀文獻，為什么這項研究能發表在Science上，做了些什么工作，有什么核心發現。

 

An alternative splicing event

evolutionary differences

 

問題：

• Comparative transcriptomic analyses revealed that vertebrate organ alternative splicing (AS) patterns diverged more rapidly than gene expression differences (4–6). 【評價指標是什么？】
• These AS differences were largely attributed to changes in the use of conserved cis-regulatory elements (4, 5).【不懂，AS跟順式作用元件有什么關系】
• 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.【大概有一點懂了】
• 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.【需要好好看他們是怎么處理的】
• The percentage of transcripts with an intron retained (PIR) was estimated for intron-retention events (10).【PSI和PIR有什么區別？】

 

 

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).【測了不同處理的cells的RNA-seq，分析了AS。組別：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【過濾標准】
• 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【調控差異不是基因表達導致的，而是可變剪切】
• 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.【一個核心結論，exon 9和RRM2的功能具有相對獨立性】

 

• 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).【開始做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.【核心結論】

 

• 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).【檢驗skipping of exon9是否會繼續降低PTBP1表達，同時激活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).【在神經系統分化過程中，Ptbp1基因表達降低，同時exon 8 skipping增強】

• 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).【兩個AS調控因子】

• 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.【跳過exon9會降低PTBP1的活性，接下來要探究在非哺乳類里面的反應】

• 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.