Post-transcriptional regulation of an intronic microrna

Post-transcriptional regulation of an intronic microrna Carl Novina Dana-Farber Cancer Institute Harvard Medical School Broad Institute of Harvard and MIT Qiagen Webinar 05-17-11

Outline 1. The biology of micrornas 2. MicroRNAs are dysregulated in cancers 3. The biology of melanomas 4. mir-211 suppresses melanoma invasion 5. Coupled splicing of melastatin and microprocessing of mir-211

Characteristics of micrornas Class of regulatory molecules. Genetically encoded 21-25 nucleotide RNAs. > 1,000 known in humans. Involved in differentiation, proliferation, apoptosis, development. High degree of conservation between species. Usually bind to the 3 -UTR of target gene with imperfectly complementary basepairing. Post-transcriptional regulation. Computationally predicted to target 1/2 of human genes. Dysfunction linked to diabetes, hypercholesterolemia, and most notably cancers.

Overview of microrna biogenesis Ding et al., Trends Biotechnol. 27:27 (2009)

micrornas have been implicated in many mechanisms of gene regulation 1. Translation repression 2. mrna cleavage 3. mrna deadenylation 4. mrna storage and surveillance in stress granules & P-bodies 5. Recycling out of P-bodies 6. mrna decapping 7. Translation activation 8. Transcription repression 9. Transcription activation 10. Others? Filipowicz et al., Nature Rev. Genetics, 9, February 2008

post-transcriptional regulation of mirna expression Heo et al., Mol. Cell 32:276 (2008) Some factors regulate pri-mirna processing to pre-mirnas: hnrnpa1 (+/- let-7), KSRP (+ let-7), p68 (+), p72 (+), p53 (+), SMADs (+). TUTase regulates pre-let-7 processing to mature let-7: oligouridylation followed by unknown nuclease degradation. GLD2 regulates mature mirna stability: polyadenylates & stabilizes mir-122a.

microrna expression profiling classifies cancers 1. Expression of many micrornas change in cancers relative to untransformed cells. 2. Some tissue-specific micrornas do not change in cancers and thus may be used to identify the tissue of origin for particular tumors. 3. Are micrornas different from other markers? -same tools to detect micrornas and modulate function - modulation may alter progression and drug sensitivities

Reduced let-7 expression correlates with poor prognosis in lung cancers Takamizawa et al., Cancer Res. 64:373 (2004) let-7: reduced in many cancers inhibits known oncogenes such as RAS increased expression inhibits cell proliferation

Multiple (especially aggressive) cancers aberrantly express Lin-28 or Lin-28B and down-regulate let-7 Viswanathan, Nat. Genet. 41:843 (2009) Mendell, Nat. Genet. 41:78 (2009)

Increased microrna expression can induce cancers He et al., Nature 435:828 (2005)

Inactivating mirnas can block cancers or cancers can become addicted to mirnas Medina et al., Nature 467:86 (2010)

Melanoma Malignant tumor of melanocytes. 160,000 new cases every year and its incidence is increasing. 48,000 deaths per year. Causes 75% of all deaths associated with skin cancer. Lethal and treatment refractory if escapes early detection. Mechanism(s) of melanocyte transformation and melanoma metastasis are unknown.

Melastatin A founding member of the transient receptor potential (TRPM1) cation channel family. Highly expressed in melanocytes and retinal pigmented epithelium. Robustly expressed in benign and dysplastic nevi and in melanomas in situ. Variably expressed in invasive melanoma and shows widespread down-regulation in melanoma metastasis. Expression correlates inversely with metastatic potential and prognosis of melanomas. Mechanism(s) by which mela-statin might suppress melanoma metastasis remain unknown.

Outline 1. What are micrornas 2. MicroRNAs are dysregulated in cancers 3. The biology of melanomas 4. mir-211 suppresses melanoma invasion 5. Coupled splicing of melastatin and microprocessing of mir-211

mir-211 is a melanocyte-specific mirna Gaur et al., Cancer Res. (2007) mir-211

Could mir-211 be responsible for tumorsuppressive activity in melanoma?

Melanomas with low mir-211 expression have high invasive potential

but slow growth rates

Expression of mir-211 and melastatin are tightly correlated: melanocytes & melanomas Levy et al., Mol. Cell (2010)

Linear regression of mir-211 and melastatin: expression in melanomas Levy et al., Mol. Cell (2010)

Decreasing mir-211 levels increases invasive activity of melanomas Levy et al., Mol. Cell (2010)

Increasing mir-211 levels decreases invasive activity of melanomas Levy et al., Mol. Cell (2010)

Decreasing mir-211 but not melastatin increases invasive activity of melanomas non-invasive invasive melanoma epidermis mir-211 mir-211 melastatin melastatin?

mirna gene architecture

Microprocessor-dependent increases in melastatin exon 6-exon 7 junctions

The RNase III activity of Drosha is required to promote splicing at a mir-211-hosting intron

5 SS recognition promotes microprocessing of and Drosha recruitment to intronic mir-211

Knockdown of 5 SS factors globally decreases intronic mirna levels

A feed-forward model of microprocessing and splicing

Conclusions Melastatin and mir-211 are coordinately reduced in melanomas. Reducing mir-211 but not melastatin expression increases melanoma invasiveness. The early steps of melstatin exon 6-exon 7 splicing and microprocessing of mir-211 are directly coupled processes. When primary transcript levels are low, splicing of host genes and microprocessing of intronic mirnas may be coupled. Splicing and microprocessing may have co-evolved, at least in the case of mirtrons.

Acknowledgements Novina Lab Bingbing Wang Steffen Schubert Howard Chen Shuqiang Li Maja Janas Carmit Levy Mehdi Khaled David Fisher Levi Garraway Jun Lu Todd Golub Dimitrios Iliopoulos Andrew Kung Sophie Pinner Anne Fletcher Shannon Turley