10. 基因转录后水平调控

Splicing of pre-mRNA occurs in a “spliceosome” an RNA-protein complex
spliceosome
(~100 proteins + 5 small RNAs)
pre-mRNA
spliced mRNA
The spliceosome is a large protein-RNA complex in which splicing of pre-mRNAs occurs.
Secondary Structure
G G HAIRPIN LOOP
A
U
U
G
BULGE C
C
G
G
A INTERNAL LOOP
U
A
STEM A
U
G
C
A
5’
G C U U DANGLING ENDS
3’
RNA “tertiary interactions”
In addition to secondary structural interactions in RNA, there are also tertiary interactions.
第一部分 RNA structure/folding
Ribozyme
Ribosome
Nobel prize 1989
Nobel prize 2009
The function of the RNA molecule depends on its folded structure
RNA functions
PARS: Parallel analysis of RNA structure
DMS: dimethyl sulphate
第二部分 mRNA transport
The Nuclear Pore Complex
NPC: all traffic between nucleus & cytoplasm in higher eukaryotes occurs via NPC
Gene Expression:
Multiple, Spatially and Temporally Distinct Steps
Carried out by Distinct Cellular Machinery
Cytoplasm
Degradation
Mature mRNA
Mature mRNA
Protein
• Restrictions:
– No knots – No close base pairs – Base pairs: A-U, C-G and G-U
Protein structures
RNA structures
Total 72000
Total <2000
Due to the limited amount of data Predicting RNA tertiary structure is almost impossible
• Splice sites are the sequences immediately surrounding the exon-intron boundaries • Splicing junctions are recognized only in the correct pairwise combinations
• Why export as a protein/DNA complex? RNAs are too big and lack the signals to interact w/ nuclear export receptors
• Specific “adaptor” proteins must first bind to the RNA and chaperone this molecule to the export receptor, which, in turn, guides the RNA across the NPC
• 转录水平 • 转录后水平 • 翻译水平调控 • 蛋白质加工水平
翻译
染色质的空间变化 转录 RNA的加工成熟
mRNA的降解 多肽链的加工修饰 蛋白质的降解
基因转录后水平调控主要方式
第一部分 mRNA structure/folding 第二部分 mRNA transport 第三部分 mRNA stability/degradation/decay 第四部分 mRNA editing 第五部分 mRNA methylation 第六部分 mRNA selective degradation 第七部分 miRNA/ncRNA (epigenetic section)
Recently discovered important new roles for RNAs
In normal cells:
• in "defense" - esp. in plants
• in normal devel, siRNAs, miRNA
• for gene therapy or to modify gene expression
基因转录后水平调控
Post-transcriptional Processing of RNA
Nucleus
DNA
Pol II transcription
Primary RNA transcript Nuclear processing Capping Splicing Polyadenylation
Structural
• e.g., rRNA, which is a major structural component of ribosomes
BUT - its role is not just structural, also:
Catalytic
RNA in the ribosome has peptidyltransferase activity
Nuclear localization sequence is required for protein nuclear import
NLS: consists of either one or two stretches of basic amino acids
Functional NLS is required for nuclear localization (mutation in NLS results in cytosolic localization)
Export
Translation
- Regulation Can Be at Several Different Levels - Dynamic Protein Association
Gene Expression: Complex Network of Coupled Interactions
Cytoplasmic filament
Cytoplasm
Cytoplasmic ring
Nucleus
Inner ring Basket Distal ring
More on the nuclear pore complex
NPC size: diameter = 120nm, 8-fold rotational symmetry, MW = 125 million daltons, composed of 50-100 different proteins called nucleoporins (Nups)
Pseudoknot
Kissing hairpins
Pseudoknot
Single-Stranded
Stem
Interior Loop
Hairpin-bulge
Bulge Loop
Hairpin loop
Junction (Multiloop)
RNA structure prediction
Two primary methods for RNA secondary structure prediction: -Co-variation analysis (comparative sequence analysis)
• RNAi • RNA aptamers
Hierarchical organization of RNA molecules
Primary structure
5’ to 3’ list of covalently linked nucleotides, named by the attached base Commonly represented by a string S over the alphabet Σ={A,C,G,U}
Maniatis, T., and Reed, R. (2002). Nature 416, 499-506.
All eukaryotic mRNAs are processed
mRNA transport
1. 5’加上m7GpppN 5 （capping戴“帽”）
Specific process for Pol II transcripts
Free Energy(E) = E(CG)+E(CG)+…..
Computing RNA secondary structure: Minimum free-energy method
• Working hypothesis:
The native secondary structure of a RNA molecule is the one with the minimum free energy