Summary for last class
- General mRNA turnover pathways
- 5’⇒3’ && 3’ ⇒ 5’
- deadenylases (PAN, CCR-NOT)
- decapping complex
- Xrn1, exosome, DcpS
- Aberrant RNA turnover pathways
- Premature stop codons: nonsense-mediated mRNA decay (NMD)
- No stop codons: non-stop mRNA decay (NSD)
- Elongation stall: no-go mRNA decay (NGD)
- Translation into the 3’-UTR: ribosome extension-mediated mRNA decay (REMD)
- Specialized mRNA turnover pathways
- ARE-mediated mRNA turnover: AU-rich elements in the 3’-UTR are bound by proteins that module the stability of mRNAs in response to regulatory signals
- Location of mRNA turnover
- P bodies: contain 5’⇒3’ mRNA turnover machinery and degrade mRNAs that are no longer available for translation
- Exosome granules: contain PARN and exosome and participate in ARE-mediated decay
The major eukaryote mRNA turnover pathway
- 5’⇒3’
- 3’⇒5’
m6A pathway and its cellular functions
- Writer
- Eraser
- Reader/Effector
Editing can change the biomedical and biophysical properties of resulting proteins
dsRNA对于细胞而言类似于病毒，会引起免疫反应
Methods to introduce A/I editing
Non-coding RNAs
80% of our genome is transcribed
but only 2% are coded for proteins.
3D structure of genome
siRNA可以是 endogenous的，也可以是exogenous的
miRNA
Small RNAs in animals
First miRNA gene: Lin-4 (Year 1993)
Second: Let-7 (Year 2000)
clone small RNAs
thousands of miRNAs are identified in eukaryotes
miRNA biogenesis pathway
miRNA: pri-miRNA⇒pre-miRNA⇒miRNA (by Dicer)
动物极少数、植物极多数中，产生的miRNA会与mRNA 3’端结合
- Translation repression
- mRNA cleavage
- mRNA deadenylation
Non-canonical miRNA biogenesis pathway
传统生化方法范式：将细胞裂解成不同的fraction⇒看看哪个fraction能够切割RNA，使用荧光标记和淬灭剂⇒使用fraction打个质谱看看什么蛋白⇒用这些做重组蛋白进行验证
miRNAs are differentially expressed in different cells or development stages.
miRNAs and diseases
Introduction of CRISPR system
Engineer pre-sgRNA to respond to miRNA/siRNAs
miRNA-inducible CRISPR platform (MICR)
CRISPR-based miRNA reporter
Biological function of miRNA
still not clear, redundancy.
miRNA全敲除细胞表型分析（miRNA genesis）⇒筛选回复缺陷表型
miR-290/302细胞周期、多能性维持
miR-290/302家族拥有脊椎动物最古老的种子序列，与促癌miR-17/20非常接近
miR302s promote heart regeneration
miRNA as biomarkers
exosomal miRNAs
Adipose-derived circulating miRNAs regulate gene expression in other tissues
Summary
- miRNAs are 20-22 nt small RNAs that post-transciptionally repress gene expression
- miRNAs are generated by a defined biogenesis pathway consists of two sequential cleavages by Dicer and Drosha
- miRNAs play important roles in animal development and diseases
- the function for most miRNAs is still not clear
- extracellular miRNAs: biomarkers and potential signaling molecules.
long noncoding RNAs
Long noncoding RNAs refer to RNA transcripts with size larger than 200 nucleotides that have no coding potential.
- small RNAs like miRNA, siRNA, piRNA, tRNA, 5s rRNA, 5.8s rRNA et alexcluded
- Other large noncoding RNAs including 18S rRNA, 30S rRNA usually not included
- Highly tissue specific
- less conserved than protein genes
- less abundant than protein genes
- promoter region is conserved
How to define noncoding
证明一个东西不存在是不可能的
- computational approaches: homology searches across large protein and domain databases
- codon substitution frequency analysis
- cutoff at <50 aa
- Ribosome profiling
Genomic localization of lncRNA
the architechture and characterization of lncRNAs
pipeline for studying lncRNA
- RNAi
- Antisense RNA
- genomic engineering
NORAD: Noncoding RNA regulation activated by DNA damage
Xist
pRNA
RNA-DNA triplex
mRNA stabilizing IncRNA-TINCR
lncRNAs serve as miRNA sponges
RIP-Seq identified hundreds of mRNAs and lncRNAs binding TRIM71
Genome Regulation by lncRNAs
ncRNA的功能保守不一定需要依赖于序列保守
lncHOME: identify “conserved” lncRNAs based on synteny and functional elements
coPARSE-lncRNAs