miRNA and Cancer

Biogenesis and function of canonical miRNAs of animals: Step 1

Transcription of a primary miRNA transcript (pri-miRNA) by RNA pol II

• transcript typically has a 5’cap

• 3’polyA tail may or may not be present, depending on timing of the next processing step

  • depends on what machinery reaches the 3’end first (microprocessor Drosha, or polyadenylation)

Biogenesis and function of canonical miRNAs of animals: Step 2

Cleavage by the microprocessor complex (Drosha +DGCR8) to form the Pre-miRNA hairpin

• Drosha is an RNAse III enzyme, these act on dsRNA

• DGCR8 is an essential cofactor, it recognizes the pri-miRNA

Drosha Cleavage

• nuclear enzyme (a class II RNase III) that acts as the primary "scissors" in microRNA (miRNA) biogenesis

  • pri-miRNA (long transcript) → pre-miRNA (hairpin ~70nt)

• pri-miRNA has a hairpin structure with the basal junction, which is a major reference point for Drosha cleavage. The other motifs also play a role in placement of the cut sites

• at least 3 of these motifs appear in 79% of human miRNAs (do not need to contain all 4 motifs for proper cleavage)

• Drosha cuts the RNA about 11 NT up from the basal junction, and about 22 NT down from the apical junction

Biogenesis and function of canonical miRNAs of animals: Step 3

Export of the pre-miRNA hairpin to the cytosol

• transport complex is formed by Exportin 5 (EXP5) plus Ran (a GTP-binding protein)

  • nucleic acids don’t have export signals, so it hitchhikes uses EXP5/Ran complex

• GTP is hydrolyzed upon transport of the pre-miRNA thru the nuclear pore. This causes release of the pre-miRNA from the transport complex into the cytosol (complex becomes inactive)

Biogenesis and function of canonical miRNAs of animals: Step 4

Dicer cleaves the loop off the pre-miRNA, leaving a miRNA duplex

Dicer

• Dicer binds the pre-miRNA in a particular orientation, based on the 3’ overhang left by Drosha

• once bound, it cleaves the hairpin at a fixed distance from the 3’end (typically 21-25 NT, depending on the species and type of Dicer protein)

• Dicer may also be bound w/ cofactors, these are other dsRNA-binding proteins

  • eg. TRBP= TAR RNA binding protein, a protein that modulates Dicer function in humans

Biogenesis and function of canonical miRNAs of animals: Step 5

miRNA duplex is then loaded onto the AGO (Argonaute) protein to form the RNA-induced silencing complex (RISC)

• assembly involves loading and unwinding the miRNA duplex

RNA-induced Silencing Complex

• multi-protein ribonucleoprotein complex that uses a small guide RNA—microRNA (miRNA) or small interfering RNA (siRNA)—to target and silence specific messenger RNAs (mRNAs)

  • core of this complex is an Argonaute (AGO) protein, which binds the guide RNA and acts as an endonuclease (or "slicer") to degrade target mRNA, thereby reducing gene expression

• the loading complex binds the dsRNA and brings it to the Ago

• one of the RNA strands is degraded the remaining RNA strand becomes the guide RNA

• the actual process of loading a dsRNA complex and activating the RISC is the same for miRNA and siRNA

Biogenesis and function of canonical miRNAs of animals: Step 6

• RNA-induced silencing complex (RISC) uses the guide RNA to bind to the target mRNA

• the complex then silences the mRNA

• if the gudie RNA is a perfect match, the target mRNA is degraded (siRNA pathway)

• if the guide RNA has some mismatch, translation of the target mRNA is suppressed (miRNA pathway)

Human Ago Proteins

• 4 different Ago proteins (Ago1 → Ago 4)

• only Ago2 is capable of cleaving perfectly matches RNAs

• all 4 Ago proteins interact w/ translational machinery and suppress translation of mRNAs. The mRNAs eventually decay

• there is no strict sorting of dsRNA types, they all work with miRNA and siRNA duplexes

Contrast w/ Drosophila Ago proteins

• Drosophila have 2 diff Ago proteins (Ago1 and Ago2)

• Ago1 preferentially loads miRNA duplexes, whereas Ago2 preferentially loads siRNA duplexes

• sorting is also guided by slight differences in the 5’NT (is it a U or a C?)

• likewise, C. elegans Ago proteins distinguish b/w miRNA and siRNA

Recall the lin-4L transcript identified. Which step of the miRNA processing pathway is this most likely to represent?

Step 2: Cleavage by the microprocessor complex (Drosha +DGCR8) to form the Pre-miRNA hairpin

Recall the lin-4S transcript identified. Which step of the miRNA processing pathway is this most likely to represent?

5) miRNA duplex is then loaded onto the AGO (Argonaute) protein to form the RNA-induced silencing complex (RISC)

How would you predict the lin-4 miRNA to regulate expression of lin-14

Repress the translation of lin-14 mRNA

Typical miRNAs

• transcribed by RNA pol II

• are transcribed from noncoding sequences outside genes or within introns

• are sometimes derived from exon sequences

• are sometimes derived from transcripts that form multiple hairpins (and multiple miRNAs)

Can miRNAs enter the hairpin processing and silencing complex thru other sources

Yes?

• don’t memorize the figure, but know that there are other RNA transcripts that can be cleaved to generate pre-miRNA hairpins or RNA duplexes, that can enter the miRNA pathway and be processed for loading onto Ago

Recall: Tumor Suppressors + Proto-Oncogenes

• Tumor suppressors: their normal function is typically to suppress cell proliferation and induce cell death. Loss of function can result in cancer

• Proto-oncogene: their normal function is to promote proliferation and to protect against cell death. Gain of function can result in cancer

• miRNAs can also act as tumor suppressors or proto-oncogenes

You identify a miRNA that suppresses cell proliferation. Which genes are more likely to have binding sites for this miRNA within their mRNA transcripts?

A. Map kinase: a protein that phosphorylates and activates other kinases in a cell signal pathway.

B. p21: A protein that binds and inhibits Cdk/cyclin complexes.

C. Wee1: A kinase that adds an inhibitory

A) Map kinases act in signal transduction pathways to promote cell proliferation

You identify a miRNA that suppresses p21, a protein that binds and inhibits Cdk/cyclin complexes. What would happen if this miRNA had a gain of function mutation?

A. Increased cell proliferation

B. Decreased cell proliferation

A

miRNA as tumor suppressor

• normal role is to suppress translation of a proto-oncogene

• a loss of function mutation could result in elevated levels of oncogene expression, and formation of tumor formation

miRNA as proto-oncogene

• normal role is to regulate the translation of a tumor suppressor gene

• a gof mutation could promote cancer by abnormal blocking expression of the tumor suppressor

Are miRNAs associated w/ human cancers?

Yes, several!

• e.g. let-7 family members

Let-7 on Cancer: Ras

• recall the role of Ras in receptor tyrosine kinase signaling (RTK) pathways; when RTK is activated, adaptor proteins help load Ras w/ GTP to activate Ras

  • active Ras then triggers downstream pathways that promote cell growth and division

  • Ras turns off when it hydrolyzes GTP → GDP

• let-7 family members negatively regulate Ras

  • if there’s a loss of let-7 → no repression of Ras → increase in cell division

Let-7 in C.elegans

• let-7 is required for cell fate determination and terminal differentiation of seam cells (specialized epithelial cells)

  • cells exit cell cycle after terminal differentiation

  • let-7 LOF mutation results in seam cells that keep cycling

Let-7 in Humans

• let-7 family members have mapped to regions that are deleted in cancers - a tumor suppressor

• overexpression of let-7 miRNA in a human lung cancer cell line inhibited its growth

Exp to Identify the Target Genes of the miRNA let-7

Strategy: computational search for C. elegans genes with sequence complementary to let-7

• revealed that miRNA let-7 binds to the 3’UTR of let-60 and represses its expression

• let-60 has complementary sites within its 3’UTR → human ortholog is RAS (a proto-oncogene)

After showing let-7 targets let-60 (Ras) in worms, does this also happen in humans? Exp

• looked at 3’UTRs from human N-RAS, K-RAS and H-RAS, w/ location and predicted duplexes formed by human let-7A

Test if let-7 suppresses translation of its target Data

Experiment:

• human liver cell carcinoma cells transfected w/ a dsRNA let-7 precursor

• human RAS protein detected w/ fluorescent antibodies

Describe the data:

• let-7 represses Ras expression (same amount of nuclei, but little fluorescence)

Test if let-7 regulates translation of RAS through through the RAS 3’UTR

Experiment:

• N-RAS (short and long forms) and K-RAS 3’UTR was fused to a luciferase reporter gene.

• Reporter constructs were transfected into HeLa cells (human cell line), and luciferase activity was normalized to the control sample (luciferase vector lacking the RAS 3’UTR)

Interpretation of Results

• let-7 suppresses expression of the luciferase reporter

• The longer N-RAS 3’UTR is subject to greater regulation