MicroRNA biogenesis and processing

Question 1

What is SHIP1?

Answer 1

Phosphatase that negatively regulates cytokine signaling via repression of PI3K pathway. Defective IFNg production
Shown to inhibit CD8+ T cell cytotoxicity (Tarasenko et al.).

Question 2

What are miRNA-155 targets?

Answer 2

cMaf, PI3K p85, SOCS1, SHIP1 –> can influence IFNg responses by T cells

Question 3

What induces miRNA-155 expression?

Answer 3

NFkB dependent factors and AP-1 downstream of BCR and TCR

Question 4

What is the origin of miRNAs?

Answer 4

Derived from ss RNAs that fold back onto themselves into stem loop structures

Question 5

What is the origin of siRNAs?

Answer 5

From ds RNA precursors that result form convergent bidirectional transcription, inverted repeat regions in structured RNA, or base pairing btw protein coding genes and pseudogene derived antisense transcripts.

Question 6

Seed region?

Answer 6

Conserved region within the 5’ terminal region of the miRNA (2-8 nt)

Question 7

Inhibitors of Drosha processing?

Answer 7

Lin28 (terminal loop –> uridylation –> degradation) - mostly sequestering of pro-miRNA
NF90+NF45 (nuclear factor)
mature miRNA
ADAR (adenosine deaminase acting on RNA) –> Tudor SN–> degradation; reduced overall stability and changes dsRNA structure
ERa inhibits helicases

Question 8

Enhancers of Drosha processing?

Answer 8

p68/p72 helicases: bind to the stem of pri-miRNA
KSRP (2-3 sequential guanidines in TL, GGG), TDR-43
hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1) (miR-18a) (sometimes neg reg) (UAGGA)
SF2/AF
SMAD (CAGAC), p53, and BRCA1 –> p68 and p72 helicases

Question 9

Inhibitors of Dicer processing?

Answer 9

Lin28 –> TUT4–> uridylation –> degradation (recognition based on primary sequence and structure of TL) or by sequestering pri-miRNA in the nucleoli (GGAG)
Lin28 –> MCPIP1 –> cleaves the loop (competition)
ADAR (adenosine deaminase action on RNA) –> Tudor SN
viral RNA factors ~800
BCDIN3D –> methylation of 5’ end

Question 10

Enhancers of Dicer processing?

Answer 10

KSRP, TDR-43 (Tar DNA binding protein 43), MBNL1

p68/p72 helicases - stabilization of Dicer

Question 11

What inhibits Exportin 5 function?

Answer 11

Viral non-coding RNAs compete for the pre-miRNA position

Bmnp-miR-1 –> targets Ran-GTP

Question 12

What is the difference btw RNAse II and III like enzymes?

Answer 12

RNAse II = single stranded RNA

RNAse III = dsRNA

Question 13

What is GW182?

Answer 13

Glycine-tryptophan repeat containing protein required for gene silencing

Question 14

What indicates potential for transcriptional regulation?

Answer 14

Presence of promoter regions that contain CpG islands, TATA box sequences, initiation elements, and histone modifications

Question 15

How does DGCR8 function?

Answer 15

Binds to the junction between single stranded and ds regions of the pri-miRNA and directs Drosha to cleave 11bp downstream of the junction –> product with 2nt 3’ overhang

Question 16

Why regulation of TLs?

Answer 16

14% of human pri-miRNAs have conserved nucleotides in their terminal loops

Question 17

KSRP

Answer 17

Its activity is modulated through phosphorylation state in response to different stimuli and provides a link btw PI3K/AKT signaling and miRNA processing

Question 18

How do mature-miRNAs affect miRNA processing?

Answer 18

Binding to specific 3’ sites to enhance processing
Inhibitory effects –> nuclear localization of mature miRNA (localization signaling contained within the mature sequence)

Question 19

Regulatory feedback loops?

Answer 19

Drosha-DGCR8 complex –> DGCR8 stabilizes the Drosha protein in the microprocessor complex, which in turn cleaves the hairpin structures embedded in the 5’ UTR of DGCR8 mRNA–> degradation of DGCR8 transcript

Ensure the optimal microprocessor concentrations in order to avoid mRNA degradation

Question 20

Export of pre-miRNA from nucleus?

Answer 20

Exportin 5 + Ran-GTP
Length of ds stem and presence of 3’ overhang needed for Exportin 5 recognition
Exp5 interacts with Dicer mRNA –> accumulation of Dicer

Question 21

Dicer cleavage?

Answer 21

Dicer alone can catalyze the cleavage but TRBP+PACT enhance the specificity ; stabilization of Dicer

TRBP phosphorylated by Erk

Dicer specifically recognizes the 5’ monophosphate

Question 22

What is the role of Ago2?

Answer 22

Ago enhances expression of endogenous miRNAs

• miRNA stabilization: phosphorylated within the RNA binding pocket –> inhibits small RNA binding
• Catalyzes an alternative pre-mirna processing event –> cleavage within the 3’ arm of the pre-miRNA–> only small RNA generated from 5’ arm is functional –> passenger strand dissociation for hairpins with high complementarity, where this might otherwise be inefficient

Question 23

Alternative miRNA biogenesis pathway?

Answer 23

Libri et al. - viruses

Question 24

SNPs to regulate miRNA biogenesis?

Answer 24

Sequence variations that originate from changes in DNA sequences or post-transcriptioanl modifications of RNA; SNP that affects strand selection and incorporation into RISC

Question 25

miRNA Editing?

Answer 25

ADAR (adenosine deaminase acting on RNA proteins) –> favorably bind to dsRNA –> can affect both pro- and pre-miRNA processing; cleavage by Tudor SN (Tudor staphylococcal nuclease); a component of RISC with ribonuclease activity specific for inosine containing dsRNA; interfere with Dicer processing; editing of mature miRNA can lead to different mRNA targets; binding alone can sometimes suffice;

Question 26

Regulation of miRNA stability?

Answer 26

Once mature miRNA incorporated into RISC generally considered very stable; half lives hours-days; regulated miRNA decay

Modifications to the 3’ end of m-miRNA –> methylation; adenylation; uridylation - nucleotidyl transferases

Adenylation at 3” end associated with both decreased and enhanced stability (GLD2 - monoadenylation)

Sequence motifs regulating miRNA stability - cis acting elements –> specificity to miRNA degradation process –>

Trans-acting factors: XRN family of enzymes: degrading the loop sequence of pre-miRNA and pri-miRNA; hPNPase –> selective down regulation in melanoma cells; enhance the stability of miRNA –> STAR (signal trunsduction and activation of RNA)

miRNAs generally outlive their targets; target recognition promotes modification (uridylation) and subsequent miRNA degradation

Question 27

How miRNAs control mRNA translation?

Answer 27

1. Degradation of mRNA sequence: siRNA = endonucleolytic cleavage of target mRNA sequence by utilizing components of miRNA machinery –
   miRNA = deadenylation of the polyA tail and recapping of the mRNA sequence
2. Blocking of initiation: miRNA binds to the RISC and targets the 3’ UTR of mRNA –> activates a sequence of events that block initiation proteins from binding to the 5’ cap of the mRNA; e.g. Ago2 contains a similar binding domain as the eukaryotic translation initiation factor 4E for binding the 5’ cap of the mRNA sequence –> binding loop that block initiation and subsequent translation
   - -> protein concentration decreases but mRNA levels remain constant
3. Translocation to processing bodies: translocation of miRNA:mRNA complexes into cytoplasmic foci: P bodies: enzymes and factors for mRNA turnover and repression of translation e.g. GW182 and Dcp1/Dcp2 (recapping enzyme); OR P-bodies may serve as storage units for mRNA (intermediate role)
4. Activation of translation: e.g. miR-369-3; posttranscriptonal changes in miRNA can alter its function from inhibitory to activating

Question 28

micro-RNA definition?

Answer 28

A class of non-coding RNAs that modulate gene expression at the post transcriptional level and are involved in regulating several aspects of inflammation

Question 29

miRNA turnover?

Answer 29

Small RNA Degrading Nuclease (SDN) proteins –> ssRNA (response to stimuli, e.g. hypoxia)

Poly(A) polymerase GLD-2 catalyzes the addition of a 3’ -terminal adenosine following Dicer cleavage and unwinding of miR-122 –> promotes stability of miRNA

Question 30

Regulation of miRNA activity?

Answer 30

Proteins binding to Ago determine the miRNA activity.

Question 31

Question to ask when studying miRNA biogenesis and processing?

Answer 31

1. Is nuclear export compromised? –> measure nuclear vs. cytoplasmic pre-miRNA. If effectively exported –> Is is a Dicer regulated step in question?
2. Is an activator or inhibitor involved as a regulatory factor?
   - Can pre-miRNA be processed with recombinant Dicer? If YES, not an activator model
   - Inhibitory factor: Addition of increasing amounts of cytoplasmic lysate extracts readily abolishes processing–> presence of an inhibitor
3. Regulatory proteins? Drosha binding assoc. proteins: Ribonucleoprotein Complex Immunoprecipitation + Gel Mobility Shift Assay : Cell lysate IP-ed with either Protein A or Protein A/G sepharose coupled coupled antibodies at 4C o/n; Wash buffers: I 150mM NaCl, II 500 mM NaCl, III LiCl, NP-40, deoxycholate, EDTA, Tris-HCl, pH8.1
4. Acquired variations in miRNA transcripts
5. SNPs
6. Is BIC transcription regulated? –> Luciferase reporter construct controlled by a BIC promoter
   - -> TXN inhibitor (actinomycin D) - since drop in pri-miRNA, conclude that increased pri-miRNA levels do not result from increased transcription
   - -> Translation inhibitor (cyclohexamide) - since increase in pri-miR –> de novo translation of short-lived decay factors contributes to keeping pri-miRNA levels down
7. Is miRNA transcription regulated by a specific factor (e.g. SHIP1) –> siRNA seq targeting SHIp1 or a scrambled seq cloned into pTRIPZ lentiviral vector; miRNA-like processing elements to express the siRNA seq under DOX regulated promoter; small molecule SHIP activator
8. What transcription factors regulate BIC transcription? Chip-Seq; designed primers flanking STAT5 binding site in the promoter –> used to detect enrichment of this seq in chromatin from cell precipitates with STAT3 and STAT5 abs , relative to a negative control
9. To confirm if an mRNA is a direct target of miRNA-155? Clone its 3’ UTR into a luciferase reporter plasmid/mutated 3’UTR; Search 3’UTRs of significantly unregulated genes in microarrays –> seed sequences
10. Can you rescue miRNA-155 by activating isolated T cell in vitro?
11. Is BIC transcript processed into miR-155? –> Transfected cells with pcDNA3.BIC = CMV promoter driving synthesis of a 417nt RNA starting at ~90nt to the 5’ site to the miRNA coding sequence –> to show that a non spliced partial copy of BIC RNA can be processed into miR-155
12. Can cell extracts alter Microprocessor mediated cleavage of pri-miRNA? –> Radiolabeled pri-miRNA substrates pre-incubated with cell extract –> subjected to processing by affinity purified Microprocessor complex –>
    Electrophoretic Gel Mobility Shift Assay: probe against radio labeled pri-miRNA –> look for band shift

• Pre-miRNA conjugated to agarose beads and incubated with whole cell extract –> affinity elute subjected to SDS-PAGE –> Colloidal staining –> Excised Bands –> Mass. Spec –> Sequencing –> Discover RNA Binding Proteins
• Kinetics of RBP expression; Co-sedimentation assay to look for binding; Flag-IP Assay.

13. Is nuclear transport regulated?
    - Export of pri-miRNA from Xenopus oocyte nuclei - radio labeled pre-miRNA and U3 and U1; export monitored at 0.5, 1, and 2 hr in the absence or presence of competitor pre-miRNA
    - Dependent on RanGTP export receptor
    - Assayed for formation of complexes between labeled pre-miRNA and Exp 5 (EMSA)

Question 32

Which microRNAs are involved in immune regulation?

Review by O’Connel

Answer 32

miR146a = negatively regulates type I IFN production in macrophages and DCs

Question 33

miRNA-155* vs miRNA-155 - Strand selection as an alternative post transcriptional regulation mechanism

Answer 33

Widely assumed that miRNA* species simply promotes the accurate processing of its miRNA partner and that miRNA* might be neutral and tolerated in vivo

Other reports suggest that miRNA* species are evolutionarily conserved and have inhibitory function equivalent to miRNA

MiRNA* can cooperate with its miRNA partner in the same physiological process e.g. type I IFN production by DCs

Factors other than the stability of both termini of the dsRNA affect the strand selection of miRNA –> e.g. amount of KHSRP and type I IFN determine amount of miRNA-155* (TLR7 activation of DCs –> type I IFN –> KHSRP –>

Question 34

DEFINITION of microRNAs

Answer 34

MicroRNAs posttranscriptionally regulate gene expression by forming imperfect base pairing with sequences in the 3’ untranslated region (3’ UTR) of genes to prevent protein accumulation by repressing translation or inducing miRNA degradation