2.circRNA的穩定性，有沒有可能是為了防止內切酶？ This high stability is presumably the result of their covalently closed ring structure protecting these molecules from exonuclease-mediated degradation.
6.如何比較circRNA在不同組織或細胞的表現量？ whether in general circRNAs are more actively produced by terminally differentiated cells, such as those in the nervous system, or whether they accumulate in non-proliferating cells owing to their high stability is unclear.
7.circRNA的表達之所以會被稀釋，和circRNA表達的測量方式有什麼關係？ circRNAs are often downregulated in cancer and in other diseases in which cell proliferation rates are high, possibly because they are diluted by proliferation before reaching steadystate levels
8.相較於直接測circRNA的增減，用circular to linear RNAs ratio有什麼好處？ inhibiting the spliceosome by depleting components of the U2 snRNP markedly increases the ratio of circular to linear RNAs
9.如果逐一地移除splicing factor，circRNA表現量的增長是指數？線性？還是？ as additive effects were observed when multiple factors were depleted, each splicing factor may play a nonredundant role in the formation of circRNAs
11.A-to-I-editing有什麼用？ dsRNA-specific ADAR enzymes prevent the activation of the innate immune system by editing adenosine to inosine in endogenous dsRNA
15.intronic lariat和ciRNA在結構上有何不同？ Lariat formation happens when the 5′ end of the intron being removed is joined to the branch-point adenosine with a 2′,5′-phosphodiester linkage
16.為什麼會有exon skipping這麼神奇的事件？ exon skipping is an event during which alternative exons are spliced out of the final mRNA product and end up contained within the excised lariat
17.為什麼會發生escape from branching？不如說，為什麼需要發生？ The hydrolysis of 2′,5′-phosphodiester bonds in intron lariats by the lariat debranching enzyme, encoded by DBR1.
21.methylation對circRNA上調或下調，分別代表什麼意義？ circRNAs were both upregulated and downregulated, indicating that the role of gene body methylation in circRNA biogenesis is dependent on the genetic context
22.除了初級結構的不同，circRNA和linear RNA在蛋白質的四個層級的結構上，有何不同？ The only region of a circular RNA (circRNA) that is distinct from the corresponding linear RNA at the primary sequence level.
25.為什麼含有intron的circRNA不會被locate到cytoplasm？ Following biogenesis, most circRNAs, with the exception of intron-containing circRNAs, are exported to the cytoplasm.
35.miR-7藉由怎樣的分子機制，召集（或留滯）miR-671的silencing complex到ciRS-7，以促進AGO2切斷miRNA（透過RNAi的機制）？ This cleavage can be enhanced by miR-7, which recruits the miR-671 silencing complex to ciRS-7 or retains it there through an as yet undefined mechanism.
37.現在如果要運輸ciRS-7，extracellular vesicles是怎麼被召集到該circRNA旁的？又運輸機制是什麼？怎麼知道要送到哪？ ciRS-7 can retain its circular nature in extracellular vesicles and function upon release in recipient cells, suggesting that export may be important for cell-to-cell communication.
41.根據kinetics，預測一個circRNA上多個RBP的關係並驗證。 circRNAs containing RNA binding protein (RBP) binding motifs may function as sponges or decoys for these proteins and indirectly regulate their functions.
44.有沒有辦法以酵素動力學的公式推導，解釋circRNA和其他分子對miRNA結合的競爭？ as highly abundant circRNAs containing many competing binding sites are more likely to have competing endogenous RNA function
46.circRNA能不能作為幹細胞分化實驗的材料？ As ciRS-7 is particularly abundant in neuronal tissues it is likely to be involved in neuronal function and differentiation,
47.要抑制miRNA下游的作用，circRNA最少需要多少binding site？ Although many other circRNAs, including circHIPK3 and circBIRC6, have subsequently been shown to have miRNA sponging properties, unlike ciRS-7, most circRNAs do not contain more miRNA binding sites than would be expected by chance.
48.為什麼circZNF91是繼ciRS-7之後，很有可能成為sponge的circRNA？如何從其序列看出來？ Based on its sequence, after ciRS-7, circZNF91 is the next most likely circRNA to act as a miRNA sponge12, and it contains 24 target sites for miR-23b-3p5.
49.不同circRNA如果target同一個miRNA，它們會不會互相合作或競爭？要怎麼驗證？ it has been hypothesized that the higher overall level of circRNAs in differentiated cells may function cooperatively to sponge numerous miRNAs
50.circRNA的生成，涉及哪些調控因子？ the introns flanking circMbl have many mbl (or MBNL1) binding sites, the binding of mbl (or MBNL1) to which facilitates the looping of the nascent RNA to promote circMbl biogenesis
51.參考Autoregulation的定義和相關paper，設計實驗驗證circRNA的autoregulatory circuit。 an autoregulatory circuit may exist in which excess mbl or MBNL1 decreases the production of its own mRNA by promoting circRNA biogenesis, and the circRNA promotes the linear splicing of the gene by tethering mbl or MBNL1
52.circANRIL如何誘發nucleolar stress？ circANRIL induces nucleolar stress and the activation of p53, which may prompt atheroprotection by removing hyperproliferative cells from atherosclerotic plaques
54.研究顯示，PES1可能受到miR‐105‐5p的調控。考慮到circRNA作為sponge的特性，circANRIL有沒有可能和miR-105-5p有交互作用？ circANRIL impairs pre-rRNA processing and ribosome biogenesis by binding to PES1, an essential 60S pre-ribosomal assembly factor
55.scaffold的機制是什麼？ Some circRNAs, such as circ-Amotl1 and circFoxo3, function as protein scaffolds to facilitate the colocalization of enzymes and their substrates.
57.FECR1如何召集TET1到FLI1？（例如釋放訊息分子） circRNA FECR1 recruits TET1 to the promoter region of FLI1, its host gene, leading to the demethylation of CpG sites and active transcription
58.G0 exit有何重要性？ This cGAMP activates the adaptor molecule STING, which stimulates the production of type I interferons causing haematopoietic stem cells to exit the G0 phase and enter an active cell cycle until they become exhausted.
59.NF90/NF110為什麼會特異結合circRNA而不是lienar RNA？ antiviral protein NF90/NF110, which preferentially binds to circRNAs rather than their linear counterparts in the cytoplasm
60.circRNA所形成的duplex為什麼能結合PKR？ The circRNAs with bound PKR are cleaved by the cytoplasmic endoribonuclease RNase L, which is activated by 2′,5′-linked oligonucleotides produced by oligoadenylate synthetase (OAS) upon binding to viral dsRNA. Subsequently, PKR molecules are released, phosphorylated and activated.
61.為什麼內源性的circRNA不能活化pattern recognition receptor？（從序列來看） foreign circRNAs can stimulate immune signalling in mammalian cells by activating the pattern recognition receptor RIG-I, whereas endogenous circRNAs do not.
"The ability of cells to distinguish between endogenous and foreign circRNAs is dependent on the intronic sequences that flank the circRNA and on the splicing machinery, rather than on the primary sequence of the circRNA."
62.RBP等splicing元件，與pattern recognition receptor的masking有關。驗證masking的機制？ Specifically, RBPs, including splicing factors, mark endogenous circRNAs as ‘self ’, potentially by masking RIG-I recognition elements in them.
63.為什麼使用RNase R得到的circRNA容易不純？ The authors of this study suggest that impurities in circRNA preparations generated using RNase R may have activated RNA sensors in previous studies,
67.circRNA的轉譯產物常是truncated version？因為它缺乏特定的functional domain。如何檢查functional domain存在與否？ circRNA-derived peptides are often truncated versions of the canonical proteins that lack essential functional domains
70.PINT87aa的結合為什麼能夠促進promoter和target的interaction？ PINT87aa binds to the RNA PAF1 complex, enhancing its interaction with the promoters of target genes by increasing binding affinity and ensuring that the PAF1 complex is in the nucleus.
71.如果功能都是調節基因表現，circRNA的轉譯產物和circRNA所吸附的miRNA之間是否有關聯？（例如都位於心臟、調控同一條pathway等等） circRNA-derived peptides can also be expressed under different conditions to the canonical protein, such as in times of cellular stress, or function in different cellular compartments to the canonical protein
82.RBP和SF如何透過與內源性circRNA結合，防止其和RIG-I的結合？ 內源性circRNA之所以無法和RIG-I結合，是因為RBPs and SFs bound to endogenous circRNAs prevent RIG-I binding and activation。 同問題61.
83.PKR要經過磷酸化，才能和viral RNA結合。那為什麼NF90/NF110不用經過磷酸化，就能結合viral dsRNA？ (6) NF90/NF110 and PKR released from circRNAs upon viral infection bind to viral dsRNA
85.不同的轉染法中，為什麼NB可以用來偵測circRNA？ Northern blotting is often used in combination with ribonuclease (RNase) R treatment to identify the band corresponding to the circular RNA (circRNA) of interest.
86.設計circRNA的primer時，需要注意哪些事情？ To detect circRNA, reverse transcription (RT)-PCR utilizes a divergent primer pair, which only amplifies the circRNA during PCR.
92.為何需要設計跨越BSJ的circRNA？ Probes span the BSJ of the circRNAs of interest and do not generate a signal when bound to a corresponding linear transcript in the reverse orientation.
96.circRNA和linear RNA來自於同一個locus，導致兩者測量會互相干擾。所以要設計方法分辨circRNA和linear RNA？ the functional characterization of circRNAs is not trivial as most circRNAs have linear counterparts that can interfere with data interpretation; conversely, abundant circRNAs may interfere with the study of linear transcripts derived from the same locus
101.如何知道circRNA產物有沒有抗體結合位？ This tag can be used to study the peptide by western blotting if no antibody against the circRNA-specific part of the peptide is available.
backsplicing is generally less efficient than linear splicing
This high stability is presumably the result of their covalently closed ring structure protecting these molecules from exonuclease-mediated degradation.
circRNAs are generally expressed at lower levels than their linear counterparts
whether in general circRNAs are more actively produced by terminally differentiated cells, such as those in the nervous system, or whether they accumulate in non-proliferating cells owing to their high stability is unclear.
circRNAs are often downregulated in cancer and in other diseases in which cell proliferation rates are high, possibly because they are diluted by proliferation before reaching steadystate levels
inhibiting the spliceosome by depleting components of the U2 snRNP markedly increases the ratio of circular to linear RNAs
as additive effects were observed when multiple factors were depleted, each splicing factor may play a nonredundant role in the formation of circRNAs
dsRNA-specific ADAR enzymes prevent the activation of the innate immune system by editing adenosine to inosine in endogenous dsRNA
要如何驗證這樣的假說？
Lariat formation happens when the 5′ end of the intron being removed is joined to the branch-point adenosine with a 2′,5′-phosphodiester linkage
exon skipping is an event during which alternative exons are spliced out of the final mRNA product and end up contained within the excised lariat
Mechanism of alternative splicing and its regulation...
https://www.sciencedirect.com/...
The hydrolysis of 2′,5′-phosphodiester bonds in intron lariats by the lariat debranching enzyme, encoded by DBR1.
Abundant circRNAs tend to have long introns flanking the exons involved in backsplicing.
是什麼樣的機制會生成較長的intron？
and they are often derived from genes with highly active promoters
circRNAs were both upregulated and downregulated, indicating that the role of gene body methylation in circRNA biogenesis is dependent on the genetic context
The only region of a circular RNA (circRNA) that is distinct from the corresponding linear RNA at the primary sequence level.
This oncogene produces the circRNA FLI1 exonic circular RNA (FECR1)
FECR1 induces the demethylation of CpG sites in cis by recruiting methylcytosine dioxygenase TET1
Following biogenesis, most circRNAs, with the exception of intron-containing circRNAs, are exported to the cytoplasm.
https://kknews.cc/...
http://europepmc.org/...
In human cells, UAP56 exports long circRNAs (>1,200 nucleotides) whereas URH49 exports short circRNAs (<400 nucleotides)
In human cells, UAP56 exports long circRNAs (>1,200 nucleotides) whereas URH49 exports short circRNAs (<400 nucleotides)
少變得更少，是否有統計意義？
This cleavage can be enhanced by miR-7, which recruits the miR-671 silencing complex to ciRS-7 or retains it there through an as yet undefined mechanism.
ciRS-7 can retain its circular nature in extracellular vesicles and function upon release in recipient cells, suggesting that export may be important for cell-to-cell communication.
which suggests that circRNAs have important non-coding functions that reflect their high stability.
circRNAs containing RNA binding protein (RBP) binding motifs may function as sponges or decoys for these proteins and indirectly regulate their functions.
act as scaffolds to mediate complex formation between specific enzymes and substrates
假設cap-dependent translation的事件A，涉及一種motif，就把它比對回linear mRNA，然後拿比對不上的部分，找尋和circRNA保守的部分。
as highly abundant circRNAs containing many competing binding sites are more likely to have competing endogenous RNA function
ciRS-7 contains more than 70 conserved binding sites for miR-7
As ciRS-7 is particularly abundant in neuronal tissues it is likely to be involved in neuronal function and differentiation,
Although many other circRNAs, including circHIPK3 and circBIRC6, have subsequently been shown to have miRNA sponging properties, unlike ciRS-7, most circRNAs do not contain more miRNA binding sites than would be expected by chance.
Based on its sequence, after ciRS-7, circZNF91 is the next most likely circRNA to act as a miRNA sponge12, and it contains 24 target sites for miR-23b-3p5.
it has been hypothesized that the higher overall level of circRNAs in differentiated cells may function cooperatively to sponge numerous miRNAs
the introns flanking circMbl have many mbl (or MBNL1) binding sites, the binding of mbl (or MBNL1) to which facilitates the looping of the nascent RNA to promote circMbl biogenesis
an autoregulatory circuit may exist in which excess mbl or MBNL1 decreases the production of its own mRNA by promoting circRNA biogenesis, and the circRNA promotes the linear splicing of the gene by tethering mbl or MBNL1
circANRIL induces nucleolar stress and the activation of p53, which may prompt atheroprotection by removing hyperproliferative cells from atherosclerotic plaques
circANRIL impairs pre-rRNA processing and ribosome biogenesis by binding to PES1, an essential 60S pre-ribosomal assembly factor
Some circRNAs, such as circ-Amotl1 and circFoxo3, function as protein scaffolds to facilitate the colocalization of enzymes and their substrates.
circRNA FECR1 recruits TET1 to the promoter region of FLI1, its host gene, leading to the demethylation of CpG sites and active transcription
This cGAMP activates the adaptor molecule STING, which stimulates the production of type I interferons causing haematopoietic stem cells to exit the G0 phase and enter an active cell cycle until they become exhausted.
antiviral protein NF90/NF110, which preferentially binds to circRNAs rather than their linear counterparts in the cytoplasm
The circRNAs with bound PKR are cleaved by the cytoplasmic endoribonuclease RNase L, which is activated by 2′,5′-linked oligonucleotides produced by oligoadenylate synthetase (OAS) upon binding to viral dsRNA. Subsequently, PKR molecules are released, phosphorylated and activated.
foreign circRNAs can stimulate immune signalling in mammalian cells by activating the pattern recognition receptor RIG-I, whereas endogenous circRNAs do not.
Specifically, RBPs, including splicing factors, mark endogenous circRNAs as ‘self ’, potentially by masking RIG-I recognition elements in them.
The authors of this study suggest that impurities in circRNA preparations generated using RNase R may have activated RNA sensors in previous studies,
Pattern recognition receptors are host receptors that recognize molecules typical for pathogens.
although thousands of circRNAs are predicted to include a putative ORF with an upstream IRES
The functional relevance of most circRNA- derived peptides is still unknown.
circRNA-derived peptides are often truncated versions of the canonical proteins that lack essential functional domains
PINT87aa binds to the RNA PAF1 complex, enhancing its interaction with the promoters of target genes by increasing binding affinity and ensuring that the PAF1 complex is in the nucleus.
circRNA-derived peptides can also be expressed under different conditions to the canonical protein, such as in times of cellular stress, or function in different cellular compartments to the canonical protein
the translation of circ-ZNF609 and of m6A-containing circRNAs was induced upon heat shock in human cells
circ-Mbl-peptide was produced and/or stabilized following starvation in D. melanogaster
（Enhancer of protein function的圖）
（f Templates for translation的圖）
Fig. 4 | circRNAs acting as miRNA or protein sponges
Binding of miR-671 to ciRS-7 triggers AGO2-dependent cleavage of ciRS-7
(4) Degradation of cleaved ciRS-7 by exonucleases
內源性circRNA之所以無法和RIG-I結合，是因為RBPs and SFs bound to endogenous circRNAs prevent RIG-I binding and activation。
同問題61.
(6) NF90/NF110 and PKR released from circRNAs upon viral infection bind to viral dsRNA
NF90/NF110 promotes circRNA biogenesis in general by stabilizing intronic RNA secondary structures and thus backsplicing.
Northern blotting is often used in combination with ribonuclease (RNase) R treatment to identify the band corresponding to the circular RNA (circRNA) of interest.
To detect circRNA, reverse transcription (RT)-PCR utilizes a divergent primer pair, which only amplifies the circRNA during PCR.
Probes span the BSJ of the circRNAs of interest and do not generate a signal
when bound to a corresponding linear transcript in the reverse orientation.
the functional characterization of circRNAs is not trivial as most circRNAs have linear counterparts that can interfere with data interpretation; conversely, abundant circRNAs may interfere with the study of linear transcripts derived from the same locus
circRNAs are often expressed at lower levels than the corresponding mRNAs
c Studying circRNA translation的圖
This tag can be used to study the peptide by western blotting if no antibody against the circRNA-specific part of the peptide is available.