1. Regulatory RNA Molecular cell biology—Chapter 9 主讲：李 芬 教授/博士
分子生物学
Molecular cell biology—Chapter 9
Regulatory RNA
主讲：李 芬 教授/博士
河南师范大学生命科学学院

2. Main Contens Intruduction Regulation by RNAs in Bacteria
Regulation by RNAs in Eukaryotes

3. Intruduction
What is regulatory RNA ?
The discovery of regulatory RNA
The importance of regulatory RNA

4. What is regulatory RNA ?
regulatory RNAs are RNA molecules that function without being translated into proteins.
Non-coding RNAs: Functional RNA molecules that are not translated into proteins .

5. The Discovery of regulatory RNA
Background:人类基因组草图带给科
学家们的困惑
The discovery of miRNAs

6. Background:人类基因组草图带 给科学家们的困惑 Background:人类基因组草图带给科 学家们的困惑
1. Human genome: 3 billion chemical base pairs
~20,500 genes
是果蝇的2倍，啤酒酵母的4倍
Human genome: only 1-2％ code for protein，
24％ is the sequence of intron,
what is another 74% for ?
3. Human genome: 90% is transcribed.
Human Genome Project information archive ( )

7. Background:人类基因组草图带 给科学家们的困惑 The data from HGP Tells us:
Most of the RNA transcribed from your genome doesn’t make protein.
The complexity of higher organisms is not decided by the number of proteins coded by its genome.

8. More and more scientists believe that Non-coding RNA functions in gene-regulatory networks that underlie the complexity of higher organisms. 8

9. 后基因组时代的基因调控：RNA 调控

10. The discovery of miRNAs
Victor Ambros
Gary Ruvkun
miRNA was first discovered in 1993 by Victor Ambros at Harvard (lin-4)
The second miRNA Let-7 was discovered in 2000 by Frank Slack as a postdoc at Harvard (Ruvkun lab)

11. The first discovered miRNA lin-4 in 1993
Ruvkun G, Wightman B, Ha I. The 20 years it took to recognize the importance of tiny RNAs. Cell Jan 23;116 (2 Suppl):S93-6.
Lee R, Feinbaum R, Ambros V. A short history of a short RNA. Cell Jan 23;116 (2 Suppl):S89-92
Thought to be an oddity not a general phenomenon

12. Breakthrough with BlastN of the second miRNA (stRNA) let-7
Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Muller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P,Davidson E, Ruvkun G. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature Nov 2;408(6808):86-9.

14. The number of the identified miRNAs is growing rapidly in recent years.
Over 5000 miRNAs have been found until the August of 2007 (The miRBase Sequence Database). These miRNAs are from primates, rodents, birds, fish, worms, flies, plants and viruses.
The data are freely available to all through the web interface at and in flatfile form from ftp://ftp.sanger.ac.uk/pub/mirbase/sequences/.

17. microRNAs had been neglected for so many years because of their small size.
The underlying reason is: people never dream that small RNAs will have important biological roles.

18. The importance of regulatory RNA
In the last few years scientists have been surprised by small nucleotide sequences, microRNAs and siRNAs (small interfering RNAs), that appear to play a role in both suppressing and promoting cancer.
“We are at a transition in our understanding of RNA,” “RNA is taking on a new role. It is a regulatory molecule.” said Phillip A. Sharp, Ph.D., Nobel laureate and Institute Professor at the Massachusetts Institute of Technology.

19. Regulatory RNA was first discovered in eukaryotes.
Does Regulatory RNA exist in prokaryotes ?

20. Regulation by RNAs in Bacteria
Small RNAs (sRNA): regulation by base pairing.
Riboswitches: regulation by metabolite-mediated
structure changes
3. Attenuation: Regulation by ribosome stop-mediated
formation of terminators

21. Small RNAs (sRNA) Targets
Regulation of translation initiation and transcription termination by altering the accessibility of RBS and the formation of terminator, respectively.
Mechanism
Regulation by base pairing with the targeted sequences on mRNAs.
Acts in trans similar to miRNA, but does not require specific machinery for action.

22. 2. Riboswitches (核糖开关) Concept
Riboswitches are regulatory RNA elements that act as direct sensors of small molecule metabolites et al to control gene transcription or translation.
According to the factor thay sense, riboswitches can be divided into four types：
Type
小分子代谢物敏感型
金属离子敏感型
环境因素敏感型
空载tRNA敏感型
riboswitche

23. Metabolite-Binding Riboswitches
Targets
Regulate translation initiation and transcription termination by altering the accessibility of RBS and the formation of terminator, respectively
Mechanism
Reside upstream of the targeted mRNA, and form specific structure to bind its small molecule ligand.
Act in cis by alteration of its own structure upon the binding of the small metabolites.

25. Figure a. Control of transcription termination by a riboswitch
代谢物
Figure b. Control of translation initiation by a riboswitch.
代谢物

26. The 2nd structures of 7 riboswitches and metabolites that they sense

27. 3. Attenuation (衰减作用) target mechanism
A premature transcription termination that switches off gene expression from amino acid biosynthetic operons after the corresponding amino acid is synthesized at an adequate level.
mechanism
Requires the participation of ribosomes that translate a leader peptide. The premature transcription termination is triggered by formation of an intrinsic terminator when ribosome read through codons of the amino acid that the operon synthesizes.

28. The TRP operon
The trp operon encodes five structural genes required for tryptophan (色胺酸) synthesis.
These genes are regulated to efficiently express only when tryptophan is limiting.
Two layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation

29. Fig 16-19
Transcription of the trp operon is prematurally stopped if the tryptophan level is not low enough, which results in the production of a leader RNA of 161 nt. (WHY?)

30. The leader RNA and leader peptide of the trp operon

31. Transcription and translation in bacteria are coupled (细菌体内的转录和翻译是偶联的)
Transcription and translation in bacteria are coupled (细菌体内的转录和翻译是偶联的). Therefore, synthesis of the leader peptide immediately follows the transcription of leader RNA.
The leader peptide contains two tryptophan codons. If the tryptophan level is very low, the ribosome will pause at these sites.
Ribosome pause at these sites alter the secondary structure of the leader RNA, which eliminates the intrinsic terminator structure and allow the successful transcription of the trp operon.

32. High Trp Low Trp Transcription of the leader RNA.
RNA Pol
Transcription of the trp operon mRNA.

33. Importance of attenuation
A typical negative feed-back regulation
Use of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan.
Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation.
Provides an example of regulation without the use of a regulatory protein, but using RNA structure instead.

34. Regulation by RNA in Eukaryotes
Molecular cell biology—Lesson 17(2)
分子生物学
Regulation by RNA in
Eukaryotes
主讲：李 芬 教授/博士
河南师范大学生命科学学院

35. Main Contens Introduction The classification of non-coding RNA
The origin, mechanism and function of regulatory RNAs in Eukaryotes

36. Epigenetics Introduction Concept
The study of meiotically and mitotically heritable changes in gene expression that are not coded for in the underlying DNA sequence.
Mechanisms
DNA cytosine methylation
histone modifications
non-coding RNAs
Human Genome Project information archive ( )

37. What is non-coding RNA ? non-coding RNAs (ncRNAs) are RNA
molecules that function without
being translated into proteins.
Non-coding RNAs: Functional RNA molecules that are not translated into proteins .

38. Versatility in form and function
non-coding RNA :
Versatility in form and function
non-coding RNA are Surprisingly numerous.
non-coding RNA have very different functions.

39. The importantce of non- coding RNA
Human Genome sequnce:
1-2% : coded for protein.
90% is transcribed.
24% : intron sequence.
What is the left 74% for?
Non-coding RNA functions in gene- regulatory networks that underlie the complexity of higher organisms.

40. Housekeeping non-coding RNA
The classification of non-coding RNA
Housekeeping non-coding RNA
miRNA
Short non-coding RNA
(20-500nt)
non-coding RNA
SiRNA
Regulatory
non-coding RNA
piRNA
Long non-coding RNA
(500-20,000nt)
lncRNA

41. The origin, mechanism and function of regulatory RNAs in Eukaryotes
Topic 1. miRNA: micro-RNA (微 RNA )
Topic 2. SiRNA：Small interfering RNA (小干扰 RNA )
Topic 3. piRNA：Piwi interacting RNA ( piwi 偶联 RNA )
Topic 4. lncRNA: long non-coding RNA (长非编码 RNA )

42. Topic I. miRNA The definition and Origin of miRNA
The biogenesis of miRNA
The action mode of miRNA
Multiple Roles of miRNA

43. 1 The definition and Origin of miRNA Size: ~21–23 nts Structure:
short hairpin loop
Origin:
Derived from cellular gene
miRNAs are small ncRNAs of nucleotides, which leads to mRNA degradation or translational inhibition of its target genes. Encoded by eukaryotic nuclear DNA and involved in multiple cellular processes via base-pairing with complementary sequences within mRNA molecules.
A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses, which functions in RNA silencing and post-transcriptional regulation of gene expression.[1][2] Encoded by eukaryoticnuclear DNA in plants and animals and by viral DNA in certain viruses whose genome is based on DNA, miRNAs function via base-pairingwith complementary sequences within mRNA molecules.

44. miRNA基因由Pol Ⅱ转录的300-1000 nts 初级转录产物 pri-miRNA
The biogenesis of miRNA 2
miRNA
的生成
Transcription
Transcription
Processing
Functional Complex assembly
Processing
Functional Complex assembly
Pol Ⅱ
miRNA genes are usually transcribed by RNA polymerase II (Pol II).
The resulting transcript is capped with a specially modified nucleotide at the 5’ end, polyadenylated with multiple adenosines (a poly(A) tail),
Pre-miRNA hairpins are exported out of the nucleus in a process involving the nucleocytoplasmic shuttler Exportin-5. 
In the cytoplasm, the pre-miRNA hairpin is cleaved by the RNase III enzyme Dicer.
miRNA基因由Pol Ⅱ转录的 nts 初级转录产物 pri-miRNA

45. 3 The action mode of miRNA
MiRNA binds to the target mNRA with limited complementarity
MiRNA binds to the target mNRA with high complementarity.
 A given miRNA may have hundreds of different mRNA targets, and a given target might be regulated by multiple miRNAs.

46. Multiple Roles of miRNA 4
(A) miRNA in development
Expression of lin-4 allows： C. elegans to proceed to the late developmental stage
miRNAs are well conserved in both plants and animals, and are thought to be a vital and evolutionarily ancient component of genetic regulation.
The first miRNA was discovered in the early 1990s.[22] However, miRNAs were not recognized as a distinct class of biological regulators until the early 2000s.[23][24][25][26][27] Since then, miRNA research has revealed different sets of miRNAs expressed in different cell types and tissues[8][28] and has revealed multiple roles for miRNAs in plant and animal development and in many other biological processes
lin-4 binds its target mRNAs by imperfect base pairing.
Victor R. Ambros

47. Multiple Roles of miRNA 4
(B) miRNA controls plant phenotypes
(控制植物表型特征)
Jaw-miRNA
控制拟南芥叶形变化
(Nature, 2003)

48. Multiple Roles of miRNA4
(C) miRNA controls the differentiation of the
hematopoietic stem cell (调控造血干细胞分化)
Three kinds of miRNA control the differentiation process of hematopoietic stem cell toward lymphocyte
3种miRNA控制造血干细胞向淋巴细胞的分化过程
( Science 2004)

49. Multiple Roles of miRNA 4
(D) miRNA In cancer
1. miRNA are cancer genes
2. miRNA are cancer classifiers
3. miRNA are cancer drivers
4. miRNA are drugs and drug targets
By measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment course based on the original tissue type.

50. Topic II. SiRNA The origin of SiRNA Biogenesis and regulation of SiRNA
Three ways of SiRNA-guided gene silencing
RNAi: mechanism and application

51. 1 The origin of SiRNA Size: Size: ~20–25 base pairs in length
double-stranded RNA molecules
Structure:
Structure:
Origin:
Origin:
SiRNA is a class of double-stranded RNA molecules, 20-25 base pairs in length.
Small interfering RNA (siRNA)：是一种小RNA分子（~21-25核苷酸），由Dicer（RNAase Ⅲ家族中对双链RNA具有特异性的酶）加工而成。SiRNA是siRISC的主要成员，激发与之互补的目标mRNA的沉默。

52. Biogenesis and regulation of SiRNA2
Biogenesis and regulation of SiRNA
dsRNA catalyzed by Dicer enzyme and formed 20-25bp long double strand SiRNA.
SiRNA was then assembled into AGO and formed functional RISC complex.
Small interfering RNA (siRNA)：是一种小RNA分子（~21-25核苷酸），由Dicer（RNAase Ⅲ家族中对双链RNA具有特异性的酶）加工而成。SiRNA是siRISC的主要成员，激发与之互补的目标mRNA的沉默。

53. Three ways of SiRNA-guided
gene silencing 3
Trigger destruction of the target mRNA. (引 起靶标mRNA的降解)
Inhibit translation of the target mRNA.
(抑制靶标mRNA的翻译)
Induce chromatin modification .(引起靶 标基因的转录沉默)

54. RNAi : mechanism and application4
RNAi : mechanism and application
What is RNAi ?
RNAi is a process in which double-stranded RNA processing to 21–23nts siRNAs triggers the degradation of a homologous mRNA.
是双链RNA（dsRNA)特异性地结合到与之序列互补 的mRNA上，导致mRNA降解，从而介导的转录水 平基因表达抑制。
RNA interference(RNAi) is a process that occurs naturally within the cytoplasm inhibiting gene expression at specific sequences. Regulation of gene expression through RNAi is possible by introducing small interfering RNAs(siRNAs), which effectively silence expression of a targeted gene. RNAi activates the RNA-induced silencing complex(RISC) containing siRNA, siRNA derived from cleaved dsRNA. The siRNA guides the RISC complex to a specific sequence on the mRNA that is cleaved by RISC and, consequently, silences those genes Read more: 

55. RNAi : mechanism and application4
RNAi : mechanism and application
The Discovery of RNAi
1998年， Andrew Fire的研究证明，在正义RNA阻断了基因表达的实验中，真正起作用的是双链RNA。这些双链RNA是体外转录正义RNA时生成的，于是提出了RNAi这个词。
RNAi是在研究秀丽新小杆线虫（C. elegans）反义RNA（antisense RNA）的过程RNAi实验图片中发现的，由dsRNA介导的同源RNA降解过程。1995年，Guo等发现注射正义RNA（sense RNA）和反义RNA均能有效并特异性地抑制秀丽新小杆线虫par-1基因的表达，该结果不能使用反义RNA技术的理论做出合理解释。直到1998年，Fire等证实Guo等发现的正义RNA抑制同源基因表达的现象是由于体外转录制备的RNA中污染了微量dsRNA而引发，并将这一现象命名为RNAi。

56. The question to be addressed is:
Why exogenous dsRNA can inhibit the expression of genes homologous to that RNA?

57. RNAi : mechanism and application4
RNAi : mechanism and application
The mechanism of RNAi
Although the origin of miNRA and SiRNA is quite different.
短siRNA片段（上面提到的A，B，C）解旋成单链RNA，然后单链RNA和蛋白结合形成RNA诱导的沉默复合物（RNA-Induced Silencing Complex， RISC ）。   RISC然后和与短siRNA互补的内源mRNA结合。如果这种结合具有特异性，那么内源mRNA就会被内切酶切割成不具翻译功能的无意义的RNA片段。但如果结合不具特异性，RISC复合物就黏附在mRNA上，从而阻止核糖体在内源mRNA上的移动和翻译，最终导致不能合成任何蛋白。
They function through similar mechanisms.

58. The heart of the RNAi mechanism4
RNAi : mechanism and application
The heart of the RNAi mechanism
1.Dicer：
RNaseIII-like multidomain ribonuclease
processes input dsRNA into small fragments
helps load its small RNA products into RISC.
Structural organization:
---A PAZ domain, binds the end
of the dsRNA
---Two RNase III domains
---Other non-conserved domains.
2. RISC
RNA induced silencing complexes : a large multiprotein complex that direct the bound siRNA or miRNA to its target and inhibit the target gene expression.

59. the key component of RISC is AGO
Ago conatins two unique domains：
PAZ ： binds to SiRNA
or miNRA
PIWI ：confers slicer
activity

60. RNAi : mechanism and application 4
The applification of RNAi
A: Gene function research 基因功能研究
For example, Applied in mammalian:
Transfect exogenous siRNA into cells
Expression of siRNA in cultured cells or animal with siRNA expression vector
B: Therapeutic Applications
a. Gene therapy 基因治疗
b. Development of new drugs 新药开发

62. Topic III. piRNA The definition of piRNA
The feature and Biogenesis of piRNA
The Functions of piRNA

63. The definition of piRNA 1
piRNA is the largest class of small non-coding RNA molecules (24-31nt) expressed in animal cells. piRNAs form RNA-protein complexes through interactions with piwi proteins.
2012年前后 piRNA 研究领域开启了令人兴奋的新时代，该领域的重要成果纷纷登上Science、Nature、Cell等顶尖杂志
After 2012 piRNA research opens up exciting new era, important achievements in this field have boarded Science, Nature, Cell and other top magazines.
转座子能在染色体不同位点间跳跃，导致基因失活甚至引发癌症。生殖细胞系中转座子的跳跃还可能导致不孕。“对于绝大多数动物来说，无法控制转座子都会最终导致物种灭绝，”
piRNA：动物基因组的大英雄

64. Feature and Biogenesis of piNRA2
Feature and Biogenesis of piNRA
Feature
Only expressed in animals.
DNA code for piRNA exists in clusters throughout the genome. ( piRNA clusters)
The expression of PiRNAs was proved to be tissue and cell specificity.
The biogenesis of piRNAs is not yet fully understood. They may derived from long single stranded precursor molecules.
Biogenesis

65. 3 The functions of piRNA in germline(生殖细胞)
silencing transposable elements in the germline.
(生殖细胞内的转座子沉默)
Participate in the regulation of spermatogenesis and reproductive (参于精子生成和生殖调控)
in non-gonadal cells (非生殖细胞)
piRNAs are endogenous small noncoding RNAs ;
Animals lacking piRNA functions show defects in gametogenesis and exhibit sterility
piRNAs have also recently been shown to have roles in multigenerational epigenetic phenomena in worms
Although piRNAs have been previously suggested to be germline-specific, recent studies have shown that piRNAs also play crucial roles in nongonadal cells.
Participate in multigenerational epigenetic phenomena In worms (参与表观遗传调控)
Destruct target mRNA, silence target gene expression (破坏目标mRNA ,沉默靶基因的表达)

66. Topic IV. lncRNA The definition and feature of LncRNA
（2002年发现）
The definition and feature of LncRNA
The origin of LncRNA
The Functions of LncRNA
LncRNA and diseases

67. introduction to lncRNA1
introduction to lncRNA
Definition：
LncRNAs are non-protein coding transcripts longer than 200 nucleotides, regulates gene expression at epigenetic, transcription and post-transcription level.
Feature:
The expression of LncRNAs has obvious specific temporal and spatial expression during development.
LncRNA broadly participates of almost all physiological and pathological processes, and is very closely associated with tumor and other diseases.
The type, quantity and functions of LncRNAs are not well understood.
Structure:
Similar to mRNA.

68. mRNA lncRNA 相 同 点 不 specific temporal and spatial expression
mRNA
lncRNA 相 同 点
specific temporal and spatial expression
Formation of secondary structure
Post-transcriptional processing, such as the
5 'cap, polyadenylation, splicing
The important role in the development and disease 不
code for proteins
regulatory function
Highly conserved between species
Poorly conserved between species
Present in the nucleus and cytoplasm
Found mainly in the nucleus
Total 20,000-24,000 mRNA
3-100 times of mRNA
The expression level: Low to High
The expression level: very low to moderate

69. Interrupted protein coding genes. Chromatin reorganization. 2
The Origin of LncRNA
Interrupted protein coding genes.
Chromatin reorganization.
The reverse-displacement of Non-coding gene during replication process.
Generating a series of local replicon.
Insert a transposase components into a gene.
蛋白编码基因的结构中断从而形成一段LncRNA
染色质重组：即两个未转录的基因与另一个独立的基因串联，从而产生 含多个外显子的LncRNA
非编码基因在复制过程中的反移位产生LncRNA
局部的复制子串联产生LncRNA
基因中插入一个转座成分而产生有功能的LncRNA

70. The function of LncRNA 3 Involved in many regulation process.
Protect protein-coding genes.
Play an important role in embryonic development.
以多种模式保护蛋白编码基因：如受到严格的功能限制以保护编码基因的ORF；或表现为基因序列的较短延伸，以保护功能域和结构
such as functional limitations are strictly to protect the ORF encoding genes; or gene sequences showed shorter extension, in order to protect functional domains and structures.
一些lncRNA实际上是某些调控RNA（如microRNA或piwi RNA）的前体
Precursors of certain regulatory RNA
(such as miRNA or piRNA)

71. LncRNA and diseases 4
LncRNA can regulate the function of its associated protein-encoding gene, so its inappropriate expression will result in many disease, such as:
Cancer (白血病、乳腺癌、肝癌、肺癌、结肠癌和前列腺癌等)
cardiovascular disease （心血管疾病）
neurological diseases （神经系统疾病）
immune-mediated diseases（免疫介导的疾病）
other relevant diseases（其它相关疾病）.
Although lncRNAs are among one of the least well-understood molecules, emerging studies have revealed that lncRNAs play critical roles in a broad range of biological processes and are associated with a number of diseases, i.e. cancer, cardiovascular disease, and neurodegeneration disease. LncRNAs are becoming critically important for the understanding of life sciences, especially diseases.
包括p15在内的许多因表观遗传机制而发生基因沉默的抑癌基因都可转录相应的LncRNA ，从而诱导相关疾病的发生
lncRNA在多种癌症中差异表达如：白血病、乳腺癌、肝癌、结肠癌和前列腺癌等
lncRNA失调还与心血管疾病、神经系统疾病和免疫介导的疾病等有关

72. Summary：MiRNA、SiRNA、piRNA
and LncRNA 种类
长度（nt） 来源
主要功能
siRNA
21-25
长双链 RNA
转录基因沉默
miRNA
21-23
含发卡结构的
pri-miRNA
piRNA
24-31
长单链前体或起始 转录
产物等多途径
生殖细胞内转座子 的沉默
lncRNA
>200
多种途径
基因组印记和 X 染色体失活等

73. THANKS