A cryo-electron microscopy structure of the microprocessor complex, showing human Drosha protein (ribonuclease III, green) and two subunits of DGCR8 (dark and light blue) interacting with and ready to cleave a primary microRNA. From PDB: 6V5B​.[1]

The microprocessor complex is a protein complex involved in the early stages of processing microRNA (miRNA) and RNA interference (RNAi) in animal cells.[2][3] The complex is minimally composed of the ribonuclease enzyme Drosha and the dimeric RNA-binding protein DGCR8 (also known as Pasha in non-human animals), and cleaves primary miRNA substrates to pre-miRNA in the cell nucleus.[4][5][6] Microprocessor is also the smaller of the two multi-protein complexes that contain human Drosha.[7]

A crystal structure of the human Drosha protein in complex with the C-terminal helices of two DGCR8 molecules (green). Drosha consists of two ribonuclease III domains (blue and orange); a double-stranded RNA binding domain (yellow); and a connector/platform domain (gray) containing two bound zinc ion (spheres). From PDB: 5B16​.[8]

Composition

The microprocessor complex consists minimally of two proteins: Drosha, a ribonuclease III enzyme; and DGCR8, a double-stranded RNA binding protein.[4][5][6] (DGCR8 is the name used in mammalian genetics, abbreviated from "DiGeorge syndrome critical region 8"; the homologous protein in model organisms such as flies and worms is called Pasha, for Partner of Drosha.) The stoichiometry of the minimal complex was at one point experimentally difficult to determine, but it has been demonstrated to be a heterotrimer of two DGCR8 proteins and one Drosha.[1][8][9][10]

In addition to the minimal catalytically active microprocessor components, other cofactors such as DEAD box RNA helicases and heterogeneous nuclear ribonucleoproteins may be present in the complex to mediate the activity of Drosha.[4] Some miRNAs are processed by microprocessor only in the presence of specific cofactors.[11]

Function

The human exportin-5 protein (red) in complex with Ran-GTP (yellow) and a pre-microRNA (green), showing two-nucleotide overhang recognition element (orange). From PDB: 3A6P​.[12]

Located in the cell nucleus, the microprocessor complex cleaves primary miRNA (pri-miRNA) into precursor miRNA (pre-miRNA).[13] Its two subunits have been determined as necessary and sufficient for the mediation of the development of miRNAs from the pri-miRNAs.[7] These molecules of around 70 nucleotides contain a stem-loop or hairpin structure. Pri-miRNA substrates can be derived either from non-coding RNA genes or from introns. In the latter case, there is evidence that the microprocessor complex interacts with the spliceosome and that the pri-miRNA processing occurs prior to splicing.[5][14]

Microprocessor cleavage of pri-miRNAs typically occurs co-transcriptionally and leaves a characteristic RNase III single-stranded overhang of 2-3 nucleotides, which serves as a recognition element for the transport protein exportin-5.[15] Pre-miRNAs are exported from the nucleus to the cytoplasm in a RanGTP-dependent manner and are further processed, typically by the endoribonuclease enzyme Dicer.[4][5][6]

Hemin allows for the increased processing of pri-miRNAs through an induced conformational change of the DGCR8 subunit, and also enhances DGCR8's binding specificity for RNA.[16] DGCR8 recognizes the junctions between hairpin structures and single-stranded RNA and serves to orient Drosha to cleave around 11 nucleotides away from the junctions, and remains in contact with the pri-miRNAs following cleavage and dissociation of Drosha.[17]

Although the large majority of miRNAs undergo processing by microprocessor, a small number of exceptions called mirtrons have been described; these are very small introns which, after splicing, have the appropriate size and stem-loop structure to serve as a pre-miRNA.[18] The processing pathways for microRNA and for exogenously derived small interfering RNA converge at the point of Dicer processing and are largely identical downstream. Broadly defined, both pathways constitute RNAi.[5][18] Microprocessor is also found to be involved in ribosomal biogenesis specifically in the removal of R-loops and activating transcription of ribosomal protein encoding genes.[19]

Regulation

Gene regulation by miRNA is widespread across many genomes – by some estimates more than 60% of human protein-coding genes are likely to be regulated by miRNA,[20] though the quality of experimental evidence for miRNA-target interactions is often weak.[21] Because processing by microprocessor is a major determinant of miRNA abundance, microprocessor itself is then an important target of regulation.

Both Drosha and DGCR8 are subject to regulation by post-translational modifications modulating stability, intracellular localization, and activity levels. Activity against particular substrates may be regulated by additional protein cofactors interacting with the microprocessor complex. The loop region of the pri-miRNA stem-loop is also a recognition element for regulatory proteins, which may up- or down-regulate microprocessor processing of the specific miRNAs they target.[11]

Microprocessor itself is auto-regulated by negative feedback through association with a pri-miRNA-like hairpin structure found in the DGCR8 mRNA, which when cleaved reduces DGCR8 expression. The structure in this case is located in an exon and is unlikely to itself function as miRNA in its own right.[11]

Evolution

Drosha shares striking structural similarity with the downstream ribonuclease Dicer, suggesting an evolutionary relationship, though Drosha and related enzymes are found only in animals while Dicer relatives are widely distributed, including among protozoans.[8] Both components of the microprocessor complex are conserved among the vast majority of metazoans with known genomes. Mnemiopsis leidyi, a ctenophore, lacks both Drosha and DGCR8 homologs, as well as recognizable miRNAs, and is the only known metazoan with no detectable genomic evidence of Drosha.[22] In plants, the miRNA biogenesis pathway is somewhat different; neither Drosha nor DGCR8 has a homolog in plant cells, where the first step in miRNA processing is usually executed by a different nuclear ribonuclease, DCL1, a homolog of Dicer.[11][23]

It has been suggested based on phylogenetic analysis that the key components of RNA interference based on exogenous substrates were present in the ancestral eukaryote, likely as an immune mechanism against viruses and transposable elements. Elaboration of this pathway for miRNA-mediated gene regulation is thought to have evolved later.[24]

Clinical significance

The involvement of miRNAs in diseases has led scientists to become more interested in the role of additional protein complexes, like microprocessor, that have the ability to influence or modulate the function and expression of miRNAs.[25] Microprocessor complex component, DGCR8, is affected through the micro-deletion of 22q11.2, a small portion of chromosome 22. This deletion causes irregular processing of miRNAs which leads to DiGeorge Syndrome.[26]

References

  1. 1 2 Partin, Alexander C.; Zhang, Kaiming; Jeong, Byung-Cheon; Herrell, Emily; Li, Shanshan; Chiu, Wah; Nam, Yunsun (May 2020). "Cryo-EM Structures of Human Drosha and DGCR8 in Complex with Primary MicroRNA". Molecular Cell. 78 (3): 411–422.e4. doi:10.1016/j.molcel.2020.02.016. PMC 7214211. PMID 32220646.
  2. ↑ Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R (November 2004). "The Microprocessor complex mediates the genesis of microRNAs". Nature. 432 (7014): 235–40. Bibcode:2004Natur.432..235G. doi:10.1038/nature03120. PMID 15531877. S2CID 4389261.
  3. ↑ Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (November 2004). "Processing of primary microRNAs by the Microprocessor complex". Nature. 432 (7014): 231–5. Bibcode:2004Natur.432..231D. doi:10.1038/nature03049. PMID 15531879. S2CID 4425505.
  4. 1 2 3 4 Siomi H, Siomi MC (May 2010). "Posttranscriptional regulation of microRNA biogenesis in animals". Molecular Cell. 38 (3): 323–32. doi:10.1016/j.molcel.2010.03.013. PMID 20471939.
  5. 1 2 3 4 5 Wilson RC, Doudna JA (2013). "Molecular mechanisms of RNA interference". Annual Review of Biophysics. 42: 217–39. doi:10.1146/annurev-biophys-083012-130404. PMC 5895182. PMID 23654304.
  6. 1 2 3 Macias S, Cordiner RA, Cáceres JF (August 2013). "Cellular functions of the microprocessor". Biochemical Society Transactions. 41 (4): 838–43. doi:10.1042/BST20130011. hdl:1842/25877. PMID 23863141.
  7. 1 2 Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R (November 2004). "The Microprocessor complex mediates the genesis of microRNAs". Nature. 432 (7014): 235–40. Bibcode:2004Natur.432..235G. doi:10.1038/nature03120. PMID 15531877. S2CID 4389261.
  8. 1 2 3 Kwon SC, Nguyen TA, Choi YG, Jo MH, Hohng S, Kim VN, Woo JS (January 2016). "Structure of Human DROSHA". Cell. 164 (1–2): 81–90. doi:10.1016/j.cell.2015.12.019. PMID 26748718.
  9. ↑ Herbert KM, Sarkar SK, Mills M, Delgado De la Herran HC, Neuman KC, Steitz JA (February 2016). "A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting". RNA. 22 (2): 175–83. doi:10.1261/rna.054684.115. PMC 4712668. PMID 26683315.
  10. ↑ Nguyen TA, Jo MH, Choi YG, Park J, Kwon SC, Hohng S, et al. (June 2015). "Functional Anatomy of the Human Microprocessor". Cell. 161 (6): 1374–87. doi:10.1016/j.cell.2015.05.010. PMID 26027739.
  11. 1 2 3 4 Ha M, Kim VN (August 2014). "Regulation of microRNA biogenesis". Nature Reviews. Molecular Cell Biology. 15 (8): 509–24. doi:10.1038/nrm3838. PMID 25027649. S2CID 205495632.
  12. ↑ Okada, Chimari; Yamashita, Eiki; Lee, Soo Jae; Shibata, Satoshi; Katahira, Jun; Nakagawa, Atsushi; Yoneda, Yoshihiro; Tsukihara, Tomitake (2009-11-27). "A High-Resolution Structure of the Pre-microRNA Nuclear Export Machinery". Science. 326 (5957): 1275–1279. Bibcode:2009Sci...326.1275O. doi:10.1126/science.1178705. PMID 19965479. S2CID 206522317.
  13. ↑ Michlewski G, Cáceres JF (January 2019). "Post-transcriptional control of miRNA biogenesis". RNA. 25 (1): 1–16. doi:10.1261/rna.068692.118. PMC 6298569. PMID 30333195.
  14. ↑ Kataoka N, Fujita M, Ohno M (June 2009). "Functional association of the Microprocessor complex with the spliceosome". Molecular and Cellular Biology. 29 (12): 3243–54. doi:10.1128/MCB.00360-09. PMC 2698730. PMID 19349299.
  15. ↑ Morlando M, Ballarino M, Gromak N, Pagano F, Bozzoni I, Proudfoot NJ (September 2008). "Primary microRNA transcripts are processed co-transcriptionally". Nature Structural & Molecular Biology. 15 (9): 902–9. doi:10.1038/nsmb.1475. PMC 6952270. PMID 19172742.
  16. ↑ Partin AC, Ngo TD, Herrell E, Jeong BC, Hon G, Nam Y (November 2017). "Heme enables proper positioning of Drosha and DGCR8 on primary microRNAs". Nature Communications. 8 (1): 1737. Bibcode:2017NatCo...8.1737P. doi:10.1038/s41467-017-01713-y. PMC 5700927. PMID 29170488.
  17. ↑ Bellemer C, Bortolin-Cavaillé ML, Schmidt U, Jensen SM, Kjems J, Bertrand E, Cavaillé J (June 2012). "Microprocessor dynamics and interactions at endogenous imprinted C19MC microRNA genes". Journal of Cell Science. 125 (Pt 11): 2709–20. doi:10.1242/jcs.100354. PMID 22393237. S2CID 19121670.
  18. 1 2 Winter J, Jung S, Keller S, Gregory RI, Diederichs S (March 2009). "Many roads to maturity: microRNA biogenesis pathways and their regulation". Nature Cell Biology. 11 (3): 228–34. doi:10.1038/ncb0309-228. PMID 19255566. S2CID 205286318.
  19. ↑ Jiang X, Prabhakar A, Van der Voorn SM, Ghatpande P, Celona B, Venkataramanan S, et al. (February 2021). "Control of ribosomal protein synthesis by the Microprocessor complex". Science Signaling. 14 (671): eabd2639. doi:10.1126/scisignal.abd2639. PMC 8012103. PMID 33622983.
  20. ↑ Friedman RC, Farh KK, Burge CB, Bartel DP (January 2009). "Most mammalian mRNAs are conserved targets of microRNAs". Genome Research. 19 (1): 92–105. doi:10.1101/gr.082701.108. PMC 2612969. PMID 18955434.
  21. ↑ Lee YJ, Kim V, Muth DC, Witwer KW (November 2015). "Validated MicroRNA Target Databases: An Evaluation". Drug Development Research. 76 (7): 389–96. doi:10.1002/ddr.21278. PMC 4777876. PMID 26286669.
  22. ↑ Maxwell EK, Ryan JF, Schnitzler CE, Browne WE, Baxevanis AD (December 2012). "MicroRNAs and essential components of the microRNA processing machinery are not encoded in the genome of the ctenophore Mnemiopsis leidyi". BMC Genomics. 13: 714. doi:10.1186/1471-2164-13-714. PMC 3563456. PMID 23256903.
  23. ↑ Axtell MJ, Westholm JO, Lai EC (2011). "Vive la différence: biogenesis and evolution of microRNAs in plants and animals". Genome Biology. 12 (4): 221. doi:10.1186/gb-2011-12-4-221. PMC 3218855. PMID 21554756.
  24. ↑ Cerutti H, Casas-Mollano JA (August 2006). "On the origin and functions of RNA-mediated silencing: from protists to man". Current Genetics. 50 (2): 81–99. doi:10.1007/s00294-006-0078-x. PMC 2583075. PMID 16691418.
  25. ↑ Beezhold KJ, Castranova V, Chen F (June 2010). "Microprocessor of microRNAs: regulation and potential for therapeutic intervention". Molecular Cancer. 9 (1): 134. doi:10.1186/1476-4598-9-134. PMC 2887798. PMID 20515486.
  26. ↑ Fénelon K, Mukai J, Xu B, Hsu PK, Drew LJ, Karayiorgou M, et al. (March 2011). "Deficiency of Dgcr8, a gene disrupted by the 22q11.2 microdeletion, results in altered short-term plasticity in the prefrontal cortex". Proceedings of the National Academy of Sciences of the United States of America. 108 (11): 4447–52. Bibcode:2011PNAS..108.4447F. doi:10.1073/pnas.1101219108. PMC 3060227. PMID 21368174.
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