miRNA and Gene Regulation

Recall from before…

• Central Dogma: DNA → transcription → RNA → translation → protein

• there are many layers of transcription regulation (e.g. PIC assembly, elongation control, transcription factors, etc.)

• but gene regulation also occurs after mRNA is transcribed; post-transcriptional regulation

  • alternative splicing, mRNA stability (5’cap/A’tail), miRNA

miRNA

• short non-coding RNA molecules (~21-22 nts)

• regulates mRNAs by RNA-RNA basepairing

  • results in translational repression and/or target dgradation

• each miRNA can target one or several mRNAs

• over 1/3 of protein-coding genes are regulated by miRNA

• many key biological processes are controlled by miRNA, such as development, cell differentiation and and diseases (from misregulation)

• over 1500 miRNA have been uncovered in humans

miRNA Processing: Step 1

Transcription

• miRNAs are transcribed by RNA Pol II and therefore contain a 5’cap and 3’ poly(A) tail

• a miRNA can be transcribed from its own gene or be part of an intron of another gene

• following transcription, the pri-miRNA will form a hairpin with a loop

  • in some cases, multiple hairpins will be formed within one pri-miRNA

miRNA Processing: Step 2

Nuclear Processing

• each hairpin is first processed by Drosha, an endoribonuclease in the nucleus that will introduce a first cleavage to form a ~70 mer pre-miRNA

miRNA Processing: Step 3

Export

• the nuclear export is mediated by Exportin-5 associated to Ran GTPase bound to GTP

miRNA Processing: Step 4

Cytoplasmic Dicing

• following export, the pre-miRNA is released to cytoplasm

• the enzyme Dicer cleaves the loop of the hairpin, leaving a short double-stranded RNA duplex

Dicer RNAse III

• class III RNAse that specifically recognizes double stranded RNA molecules with a 3’ overhang

• endonuclease active sites are placed 6.5nm from the RNA end recognition site (~21 nts long)

  • binding to the backbone ensures processing in a non specific manner

  • one enzyme can process multiple different pre-miRNA hairpins

miRNA Targeting: Step 1

• following the formation of the short 22 nts duplex, the passenger strand is evicted by a helicase

• the complement strand is usually kept and loaded into the RISC complex

miRNA Targeting: Step 2

• mature miRNA is loaded on RIS (RNA induced silencing complex; a multisubunit protein complex)

miRNA Targeting: Step 3

• with the help of RISC, the miRNA will then hybridize with the targeted mRNA (often at 3’UTR)

• a seed sequence (conserved 7-mer) in the 5’ region of the miRNA typically aligns with the complementary regions

• if the sequence has partial complementary with mRNA, the mRNA will remain stable but translation will be inhibited (in most cases)

• if the sequence has near perfect complementarity, the miRNA will induce mRNA cleavage degradation

miRNA Targeting Figure

How does miRNA Regulate mRNA Translation

• can repress translation at the initiation stage by blocking the cap recognition site or stage of recruiting 60s ribosome subunit

• can block mRNA circularization

• ribosome dropoff

• degradation by cap removal and/or tail removal

• 30% of human proteins is regulated by miRNA

RNAi technology

• scientists can co-opt miRNA tech to perform targeted knock down of specific mRNAs using the base-pairing strategies (RNA interference)

RNAi Technology Steps

• protein of interest

• design or order siRNA/shRNA

  • targets mRNA

  • siRNA is already double-stranded; shRNA are incorporated into plasmid to be delivered into the cell

  • shRNA has to be transcribed and processed first in order to do its function, but is more stable for longer silencing

• transfect cells

• verify that the mRNA or protein of interest is downregulated

• study protein of interest

Relationship between mRNA and Protein is not 1:1 DATA (Was hinted at to be on exam!)

Total RNA vs protein in human brain samples

• y-axis: protein produced per amount of mRNA

• There is a positive correlation (r = 0.56) → more mRNA generally = more protein

• However, r ≠ 1, so mRNA levels do not perfectly predict protein levels

Interpretation of points:

• On the line of best fit: expected protein from mRNA level

• Below the line: less protein than expected → post-transcriptional repression (reduced translation)

• Above the line: more protein than expected → enhanced translation

Relationship between mRNA and Protein is not 1:1 DATA

Correlation between RNA and Protein across diff studies in diff species

Relationship between mRNA and protein is gene specific

• Each dot = one tissue (same gene across different tissues)

• Correlation (R) shows how well mRNA levels predict protein levels

Key interpretations:

• Low R value: weak mRNA–protein relationship; greater contribution from post-transcriptional regulation

• High R value: strong mRNA–protein relationship; gene expression mainly controlled at the transcriptional level

Examples:

• RPL12: low correlation → strong post-transcriptional regulation

• SORD: positive/high correlation → mainly transcriptional control

Relationship between mRNA and protein is gene specific

• correlation for all genes across tissues; certain gene classes have higher RNA:protein correlation than others

miRNA Targeting: Thermodynamic assymmetry rule

• the strand whose 5’end is less stable (more AU pairs, fewer GC pairs) is preferentially selected as the guide strand (easier to denature)

• wy? the Ago protein RISC subunit anchors the 5’ phosphate of the guide strand

• if the 5’ end of a strand is loose due to weak AU bonding, it is much easier for the Ago protein to grab that end and begin unwinding the duplex

miRNA vs siRNA vs shRNA