1. “Gene Expression Regulation”
Lesson 5
Dr. Daniela Barilà
Sector H floor 0

2. Genes for RNAs: NON CODING RNAs
Many genes are defined non coding as they do not code for a protein but are trascribed into non coding RNA (ncRNA)
About 85% of euchromatic genome is probably transcribed
About 70% of human genome is transcribed by both helixes
Similarly to proteins and mRNAs, ncRNAs are produced as precursors that undergo several processing events to become “functional”

3. …At the beginning
ncRNA: several RNAs required to support protein production : rRNAs,tRNAs
…Then
ncRNA include also a series of RNAs involved in mRNA processing: splicing, processing of rRNA e tRNA precursors, mRNA maturation)
ncRNA involved in the inactivtion of chr X, imprinting, telomers DNA synthesis

4. Nowadays
Thousands of ncRNA: the expression of several of these ncRNAs is regulated during development or is regulated in a tissue specific manner. For this reason ncRNAs play a role in many genetic disorders and in cancer.
MicroRNA (miRNA)
RNA that bind piwi (piRNA)
Small endogenous interfering RNAs (endo-siRNA)
Regulatory Long non coding RNAs (longncRNA)

5. GENES FOR RNA
Protein synthesis: mRNA, tRNA, rRNA
RNA processing
Splicing: snRNA
Cleavage: 1) pre-tRNA: RNAase P; 2)pre-rRNA :RNAase MRP;
Base modification: 1) rRNA: snoRNA; 2) snRNA: scaRNA
DNA synthesis
TERC, RNAY, RNAase MRP
Gene Regulation
General Regulators of Transcription: snRNA U1, snRNA U2,RNA SRA 1, RNA 7SK
Gene Silencing: miRNA, endo-siRNA
Epigenetic regulation: XIST, H19, HBII-85 snoRNA
Long non coding RNAs: HOTAIR, TSIX, AIR
Transposon control: piRNA and endo-siRNA

8. Small RNAs: Regulators and Guardians of the Genome:
epigenetic regulation of gene expression
Andrew Fire and Craig Mello 1998: discovery in Caenorhabditis elegans that genes
could be silenced by RNA interference.
2006: The Nobel Prize for Physiology or Medicine “for their discovery of RNA
Interference - gene silencing by double-stranded RNA”
Andrew Z. Fire (1959),
Standford University,
School of Medicine, Standford, CA, USA
Craig C. Mello (1960),
University of Massachusettes
Medical School , Worcester, MA, USA

9. Small RNAs function in several pathways
19-31 nucleotide long, behave as sequence -specific triggers for:
mRNA degradation
translation repression
heterochromatin formation
transposon control

10. Nomenclature for miRNA
microRNAs o miR are indicated by a sequential number that reflects the order in which they were discovered
Orthologues are indicated by the same number
If there are more copies of the same miR on different chromosomes, these are labelled as follows:
a second numer: if they share the same sequence
a letter if the sequences are not fully conserved
Es: miR 133a-1; miR 133a-2; miR 133b

11. Nuclear steps miRNA genomic structure and biogenesis
Genes for miRNA are located in all chromosomes and half of all miRNA
are identified in clusters that can be transcribed as polycistronic primary transcripts.
Genes for miRNA are found in introns of non-coding or coding genes and in exons of non-coding genes.
Genes for miRNA are transcribed by RNA pol II as precursor molecules with 5’m7G capping structures and 3’ poly-A tails.
These long primary transcripts of miRNA genes (pri-miRNA) are subsequently cleaved by the complex Drosha-DGCR8 (RNASEN nell’uomo) to produce a stem-loop structured precursor of about 70 nt (pre-miRNA), with a 5’ phosphate and a 2nt 3’ overhang.
After the first processing step in the nucleus, pre-miRNA with a short hairpin structure are recognized by the nuclear export factor, Exportin-5 which along with Ran-GTP-binding protein delivers pre-miRNA to the cytoplasm.

12. Drosha (RNASEN nell’uomo): RNAsi III family endonuclease, containing 2 RNAIII domain and a ds RNA-binding domain.
DGCR8 (Pasha): dsRNA-binding protein (dsRBP) that acts as a ruler to measure the cleavage site at the 11-nt position from the base of the stem structure.

13. Cytoplasmic steps miRNA genomic structure and biogenesis
Upon export, pre-miRNAs are then processed by DICER, a cytoplasmic RNAase III that chops dsRNAs into about 22-nt duplexes of mature miRNA and miRNA passenger strand, with 2-nt overhangs at the 3’ termini.
Dicer-interacting proteins are important in the next stage where ds miRNAs are unwound and loaded onto the effector complex, miRNA-induced silencing complex (miRISC).
Only one strand is loaded on the RISC (the one with less stability at 5’ end).
RISC components: Dicer, TRBP, PACT, Gemin 3, Argonaute proteins.
Ago2 exhibits endonuclease activity to slice complementary RNA sequences between positions 10 and 11 from the 5’ end of guide strand RNA.

14. Human miRNA synthesis
Nucleus
human miRNA synthesis: miRNA are transcribed by RNA pol II; miRNA have 5’ CAP and 3’ polyA. The precusor of miRNA stem-loop structure processed by ribonucleases compexes.
Nuclear complex: RNASEN complex(homolog of Drosha); cleavage of pri-miRNA and release of a stem loop structure named pre-miRNA.
Cytoplasm: pre-miRNA is exported in the cytoplasm where it is processed by DICER complex. The product of this process is a duplex of RNA. This duplex is bound by ARGONAUTE that has the ability to trigger the degradation of the passenger helix .
CAP
RNASEN complex
Cytoplasm
DICER complex
ARGONAUTE complex

15. human miR-26a1 synthesis CAP Stem-loop RNASEN DICER miRNA duplex
ARGONAUTE

19. Human miRNA gene structure
CAP
A: Transcripts devoted to miRNA production
CAP
CAP
B: miRNA trascribed from an exon ncRNA (up) or from an intron ncRNA (down)
CAP
C: miRNA transcribed from 3’UTR (up) or from an introne coding gene (down)
CAP
CAP
Exonic ncRNA
Intronic ncRNA
Hairpin
RNA coding exon

20. Mechanisms of miRNA-mediated gene regulation
miRNA-target RNA interaction: imperfect base pairing (bulge structures due
to mismatches with its target sequence.
The sequence specificity for target recognition by the guide miRNA strand is
determined by nucleotides 2-8 of its 5’ region, referred to as the “seed sequence”.
Therefore a single miRNA can target multiple mRNAs
Indeed a computational analysis of the complementarity between 7 nt of the miRNA
seed sequence and 3’ UTRs of four genome databases predicts that more than
1/3 of human protein-coding genes are regulated by miRNA.
Endogenous miRISC recognizes the 3’ UTR of its target mRNA:
HOW DOES IT REPRESS GENE EXPRESSION?
Repressing translation
Accelerating mRNA degradation
3) Sequestrating mRNA to storage compartments

21. Gene silencing by miRNAs

22. Mechanisms of miRNA-mediated translation repression
1) miRNA represses protein synthesis after its initiation
2) miRNA represses protein translation at the initiation step
3) miRNA trigger direct target RNA destabilization
Given the complexity of miRISC structure and function, it is tempting to propose that
these mechanisms may take place simultaneously and perhaps synergistically

23. Mechanisms of miRNA-mediated translation repression

24. P-body
mRNA P-bodieas, also known as DCP or GW bodies, contain non-translating mRNA and proteins involved in mRNA remodeling, decapping, translational repression, and 5’ to 3’ exonulcease activity.
Localization of miRISC and target mRNAs at P-bodies is a consequence of
translational repression rather than the trigger for gene silencing.

25. Small Interfering RNAs (siRNAs)
The RNAi phenomenon was first discovered when introduction of long ds RNAs into C. elegans triggered silencing of targeted genes (Fire et al., 1998)
Like endogenous miRNAs, long dsRNAs are processed by the Dicer-TRBP-PACT
complex. This processing step creates RNA with 2-nt overhangs at their 3’ ends and phosphate groups at their 5’ termini.
Since Dicer can use linear dsRNA or hairpin RNA substrates, DNA vectors that express hairpin RNAs have been commonly used to induce RNAi. Alternatively, the post Dicer cleavage products, siRNAs, can be exogenously introduced in the cells.
The antisense strand is the guide strand that will be used to target the mRNA
Subsequent to Dicer processing, the nt guide strand of duplex siRNA is loaded into Ago2 to form the effector siRISC. Ago 2 is the endonuclease responsible for the cleavage activity of siRISC. Perfect base pairing: siRisc cleaves its target nt from 5’ of the guide strand and the complex is recycled.

26. Small Interfering RNAs (siRNAs)

27. siRNA design
Several design programs available (also from many companies)
Two main requirements:
Specificity
2) Efficiency
Strand selection has been shown to be determined by the thermodynamic asymmetry of the siRNA duplex: the strand that is less thermodynamically stable at its 5’ end is more easily loaded into si RISC as guide strand.

28. siRNA design (19 nt)
Several design programs available (also from many companies)
I: G/C content 30% < 52% 1
II: A/U rich (better > 3) position
III: Lack of internal repeats (low Tm) 1
IV: A in position
V: A in position
VI: U in position
VII: Absence of G/C in position
VIII: Absence of G in position 13
Because Dicer processing enhances siRNA incorporation into siRISC
27-mer siRNA or 29-mer shRNA are becoming commonly used to increase
siRNA efficiency

29. siRNA delivery
IN VITRO :
several liposome like strategies (Lipofectamine, Fugene and many more)
Electroporation derived approaches (Amaxa)
IN VIVO (Therapeutic approaches) :
Aterocollagen
Cholesterol-conjugated siRNAcan silence gene expression in vivo
HDL and LDL conjugatesd particles.
Delivery: local or systematic delivery

31. Therapeutic potential of RNA interference against cancer

32. Therapeutic potential of RNA interference for neurological disorders

34. shRNA
Mammalian expression vectors:
1) possibility of stable expression
2) easier delivery in vitro
3)RNAi libraries

35. The vector uses the polymerase III H1-RNA gene promoter, as it produces a small RNA
Transcript lacking polyA and has a well designed start of transcription and a
termination signal consisting in 5T. The cleavage is after the second U (3’ overhanging U)

36. The Rnai Consortium (TRC)
Enabling large scale loss-of-function screens through the development of genome-scale
RNAi libraries and methodologies for their use.
> lentiviral clones targeting genes

37. Genome-scale loss-of function screening with lentiviral RNAi library

40. miRNA and cancer
Calin et al reported that miR-15-a and miR-16-1 are located on lucus 13q14, a site
Frequently delated in B-cell chronic lymphocytic leukemia.
Since their discovery researchers have identified abnormal expression of miRNA in
Other malignancies, including lymphomas, colorectal carcinoma, breast cancer, thyroid cancer and hepatocellular carcinomas.
Mapping efforts have revealed that many miRNAs are located in fragile regions of
the genome and several miRNAs located in these regions often have decreased
expression in cancer.
Depending on the target mRNA, miRNAs can act as either tumour suppressor
or oncogenes.; Furthermore many miRNAs expression is regulated by proteins
that participate to modulate cell cycle and growth and are part of their targets.

41. miRNA discovery and validation of regulation
Bioinformatic tools can be used to predict a potential miRNA and the binding site of
Its target mRNA.
Sequencing studies identified more than 2500 human miRNA: only a fraction of these miRNAs have been experimentallyconfirmed to regulate their predicted mRNA targets
Once a miRNA has been identified as a potential regulator of a gene, there are at least
2 in vitro experiments required to validate the finding.
Validate the miRNA binding to the predicted 3’UTR
Validate miRNA expression effect of the endogenous target mRNA expression

42. The Competing Endogenous RNA (ceRNA) hypothesis

45. PTEN tumor suppressor vs
PTENP1 pseudogene
Loss of PTENP1 in cancer results in PTEN silencing