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THE SMALL RNAs WORLD - Coggle Diagram

- - - - mRNA Degrading Complex
        
        DICER
        
        enzyme that cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short ds RNA fragements called
        small interfering RNAs and microRNAs
        
        facilitates the activation of the RNA-induced silencing complex (RISC)
        
        RISC has a catalytic component ⇾ Argonaute (degrates target mRNAs)
      - miRNAs are encoded by miR genes.
        They silence genes mainly by translational repression
      - siRNA mediate gene silencing on a transcriptional and post transcriptional level
- - - - mi156 ➜ keep the juvenile phase
        ⁍ binds to the 3'UTR of SLP (TF that pay an important role in growth and development)
        ⁍ high in the juvenile phase
        → then the level start to decrease because promoter activity goes down and consequently the target level rises (degradation)
        ⁍ if you take away the target in the SPL, the level increses very much and the plant pass to the adult phase very quickly
        
        example of regulatory cascade
      - miR172 controls the flowering (so the next step)
        →an overexpression bings to early flowering
        ⁍ SPL directly activate the expression of the miR172 gene by binding to its promoter
        ⁍ mi172 repress AP2 activity (that repress the flowering)
    - - Lin-14 has different binding sites for Lin-4.
        
        With a Lin-4 loss of funciton mutant, Lin-14 expression stay high because is not regulated anymore
    - - they can move though the cells and create spatial gradient of messanger level
        
        PHB > expressed only in the upper side of the leaves, but if we have a gain of function mutant it becomes symmetrical
        
        mi166 regulates the PHB expression
        ➡︎ The targets is expressed everywhere but the presence of miRNA prevents the expression in some regions
      - example
        
        how can a plant communicate the needs for more nutrient in the upper region?
        
        mi399
        
        targets a pathway of E2 involved in the phosphate accumulation factor
        
        ￭ mi399 expression ⇾ induced by phospahte starvation (if you overexpress them you have a overaccumulation of phosphate)
        ￭ E2 factor is regulated by mi399, infact it controls the phosphate accumulation regulatory process
        ￭ pho2 is the E2 loss-of-function mutation (if the mutant is only in the upper part you don't have phoophate accumulation and the E2 factor is active only in the roots)
        ￭ mi399 can move form the roots to the shoot where it can promote thre phosophate accumulaiton
        ￭ mi399 also partillay anneals with IPS1➜ there is no degradation but sequenstration, so mi399 cannot do its activity
- - - - uses lambda (phage) recombinaiton system to facilitate the transfer of heterologous DNA sequences between vectors.
      - based on the site-specific recombination system used by phage I to integrate its DNA in the E.Coli chromosome :
        
        ➜both organism have specific recombination sites, attP and attB and it is catalyzed by two enzymes Int and IHF
      - the process is reversible
      - goal ➡︎ make an expression clone
      - 2 step process:
        
        Cloning one gene of interest into the Entry Vecor using the BP reaction
        
        Subcloning the gene of interest from the Entry Clone to a destination vector using LR reaction producing the Expression clone
      - advantages⇾
        
        fast
        
        no use of restriction enzymes
        
        once you have made an entry clone the gene of interest can be easily sub-cloned into a wide variety of destination vectors using the LR reaction
    - - to design a functioning artificial miRNA you subimit your gene and you get to design a primer. The primers are used for a PCR which amplifies an endogenous miRNA from a vector
      - useful to silence tandem genes