RNA Seminar SS 2007

1. RNA Seminar SS 2007 Regulation of miRNA production Oliver Kuhnert

2. Outline • Introduction • What is miRNA? • miRNA production • Examples for Regulation of miRNA production • Abstract • Sources

3. Introduction • research in miRNA acquired an increasing importance • hundreds of miRNA gene´s are identified • 300 miRNAs have been validated in the human genome these miRNAs have essential functions in mammalian biology, for example a few miRNA genes have been functionally linked to specific cellular pathways

4. 2. What is miRNA? (1) • microRNAs (miRNA) short (21-23 nt long), single stranded, endogenous RNA molecules • post-transcriptionally regulate gene expression either mediate translational repression or direct mRNA cleavage by binding complementary sequences in the 3 prime UTR of mRNA • noncoding RNA these miRNAs are encoded by genes which are transcribed from DNA but not translated into a protein • encoded in the genome of animals and plants

5. 3. miRNA production (1) • Biogenesis of miRNA is a 2 step process • The first step is in the nucleus: • miRNA gene´s are transcribed by RNA Pol II • results in the production of monocistronic, dicistronic or polycistronic precursors named primary miRNA (pri-miRNA) • pri-miRNAs have 5´ 7-methylguanosine (m7) caps and 3´poly(a) tails like “normal” mRNA

6. 3. miRNA production (2)

7. 3. miRNA production (3) • The second step is in the cytoplasm

8. 3. miRNA production (4) • the miRNA guide strand is then selected for incorporation into the miRNA-containing RNA-induced silencing complex (miRISC) • these guide strand is selected by the argonaute protein  mature miRNA or miRNP (Ribonucleoprotein, all mature miRNA exists as RNPs in the cell)

9. Examples for Regulation of miRNA production 1. Paper: Post-transcriptional regulation of miRNA expression; RNA (2006), 12:1161-1167 • They shown by Northern Blots and in situ hybridization experiments that the expression of mammalian miRNAs can be regulated at the post-transcriptional level • They use miR-138 which is spatially restricted to distinct cell types, while its precursor (pre-miR-138-2), is ubiquitously expressed throughout all tissues analyzed • Pre-miR-138-2 is exported from the nucleus to the cytoplasm  cleavage of this pre-miRNA by Dicer is restricted to certain tissues and cell types

10. 2 different precursors, pre-miR-138-1 and pre-miR-138-2, which are encoded on different chromosomal loci A: Northern Blots of miR-138, miR-9 and miR-124a Northern Blot; P2 = pre-miR-138-2 P1= pre-miR-138-1 M = mature miRNA

11. Afterwards they performing in situ hybridizations with 3´DIG-labeled LNA oligonucleotide probes to investigate the overall distribution of miR-138 and its precursor A B (B) Northern Blot (A) In situ hybridization experiment on cryo-sections of E17 mouse embryos using LNA-modified probes that recognize mature miR- 138

12. Then they transfected HeLa cells transiently with plasmids encoding either pre-miR124a or pre-miR-138-2 in order to analize pre-miRNA processing in living cells A B (A) Northern blot; conversion of pre-miR-124a into mature miR-124a (B) Northern blot; converion of pre-miR-138-2 into mature miR-138-2

13. 2. Paper: Extensive post-transcriptional regulation of miRNAs and its implication for cancer; Genes&Development (2006) They present, that a large fraction of miRNAs is regulated during the Drosha processing step, and this regulation has a major impact on miRNA expression during embryonic development and in cancer

14. during mouse development at 10.5 d of gestation you can see a massive induction of Let-7 family miRNAs • They exemplified this by using Let-7g • processing of this primary transcript by Drosha yields a 79nt stem-loop precursor • further processing by Dicer leads to the 21-nt mature species • they analyzed expression of all these three molecular forms by northern blotting

15. Results of Northern Blots: A (A) Northern Blot; analysis of the molecular species of Let-7g. 18S rRNA and U6 snRNA were used for loading controls for pri-miRNA and pre-miRNA/mature

16. Expression level of primary transcript nearly constant and do not match with the mature miRNA RT-PCR analysis of the let-7g primary transcript and mature species

17. Why? They think that processing of Let-7 is blocked at the Drosha step, release of the block would enable production of mature Let-7g • developmental function of Let-7 family members in mammals is not known • They investigate expression levels of other miRNAs in mouse • In most cases the miRNAs exhibit regulated Drosha processing

18. Their data´s suggest that differentiation events that occur during embryonic development activate Drosha processing of specific miRNAs • They directly tested this with P19 cell line, which can be differentiated in culture into multiple cell types • Result: embryonic miRNAs, such as the miR-290-295 cluster, are down regulated during differentiation • In contrast, miRNAs that are up-regulated during the mouse development, including the let-7 members, are increased upon differentiation

19. as in embryonic development, the increases in Let-7 miRNAs are not coupled to transcription of the pri-miRNA (see in Fig. below)

20. in cancer cells a widespread alteration in miRNA expression was observed some miRNAs are elevated, most have significantly reduced expression • because of the data they think that these are a consequence of Drosha processing block

21. they checked these, the result was that the tumor cells had overall reduction of miRNA expression levels (see below) • Expression map: • yellow indicates increased expression; blue decreased expression • Tumor samples are in red; normal tissues black

22. Abstract • differential processing of precursor miRNAs into mature miRNAs leads to tissue and developmental-specific miRNA expression in mammals • unprocessed miRNA precursor might play a different role BUT: why produce the cell a lot of precursor miRNA in such a energy wasteful way??

23. Abstract • such regulation allows higher stringency, but also an opportunity for quick regulation precursor form of mature miRNA is already expressed and the cell can quickly produce the mature miRNA by modifying the regulator • this could be very important for fast responses like coupling neuronal processes in time

24. Abstract • Transcription of the pri-miRNA can be regulated, for example by binding of specific proteins to the miRNA promoter and causes so the transcriptional repression of these miRNA • Processing at the Dicer step can be delayed or inhibited • possible that Drosha and DGCR8 are regulated by post-translational modifications or maybe additional regulatory binding proteins are required for specific miRNA processing  The regulation of the miRNAs are a multistep process

25. Sources • Extensive post-transcriptional regulation of miRNAs and its implication for cancer; Genes&Development (2006) • Post-transcriptional regulation of miRNA expression; RNA (2006), 12:1161-1167 • MicroRNAs: expression, avoidance and subversion by vertebrate viruses; Nature Reviews Microbiology 4, 651-659 (2006) • MicroRNA Biogenesis and Cancer; Cancer Res 2005; 65(9): 3509-12 • www.wikipedia.org/wiki/MicroRNA • www.ambion.com

26. Thanx for your attention!