miRNA biogenesis: Biological impact in the development of cancer

microRNAs (miRNAs) are non coding RNAs with different biological functions and pathological implications. Given their role as post-transcriptional gene expression regulators, they are involved in several important physiological processes like development, cell differentiation and cell signaling. miRNAs act as modulators of gene expression programs in different diseases, particularly in cancer, where they act through the repression of genes which are critical for carcinogenesis. The expression level of mature miRNAs is the result of a fine mechanism of biogenesis, carried out by different enzymatic complexes that exert their function at transcriptional and post-transcriptional levels. In this review, we will focus our discussion on the alterations in the miRNA biogenesis machinery, and its impact on the establishment and development of cancer programs.

[1]  Z. Mourelatos,et al.  A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. , 2005, Genes & development.

[2]  Wenlin Huang,et al.  miR-125b is methylated and functions as a tumor suppressor by regulating the ETS1 proto-oncogene in human invasive breast cancer. , 2011, Cancer research.

[3]  J. Yong,et al.  Ars 2 Links the Nuclear Cap-Binding Complex to RNA Interference and Cell Proliferation , 2010 .

[4]  Huey-Jen Lin,et al.  Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma development. , 2010, Cancer cell.

[5]  L. Smirnova,et al.  A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment , 2008, Nature Cell Biology.

[6]  H. Nakayama,et al.  A low Dicer expression is associated with resistance to 5-FU-based chemoradiotherapy and a shorter overall survival in patients with oral squamous cell carcinoma. , 2014, Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology.

[7]  D. Haber,et al.  Dual Role for Argonautes in MicroRNA Processing and Posttranscriptional Regulation of MicroRNA Expression , 2007, Cell.

[8]  Xiaoling Li,et al.  The microRNA-processing enzymes: Drosha and Dicer can predict prognosis of nasopharyngeal carcinoma , 2011, Journal of Cancer Research and Clinical Oncology.

[9]  Xi Li,et al.  Polymorphisms in microRNA-related genes are associated with survival of patients with T-cell lymphoma. , 2014, The oncologist.

[10]  A. Hata,et al.  SMAD proteins control DROSHA-mediated microRNA maturation , 2008, Nature.

[11]  Edwin Sandanaraj,et al.  Attenuated adenosine-to-inosine editing of microRNA-376a* promotes invasiveness of glioblastoma cells. , 2012, The Journal of clinical investigation.

[12]  B. Davidson,et al.  RNA polymerase III transcribes human microRNAs , 2006, Nature Structural &Molecular Biology.

[13]  L. Aaltonen,et al.  A TARBP 2 mutation in human cancer impairs microRNA processing and DICER 1 function , 2009 .

[14]  A. Patócs.  TGF-[beta] signaling and the microRNA machinery , 2013 .

[15]  M. Esteller,et al.  Rolling the dice to discover the role of DICER in tumorigenesis. , 2012, Cancer cell.

[16]  S. Lowe,et al.  A microRNA polycistron as a potential human oncogene , 2005, Nature.

[17]  M. Gaestel,et al.  p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication , 2010, Proceedings of the National Academy of Sciences.

[18]  Kathryn A. O’Donnell,et al.  c-Myc-regulated microRNAs modulate E2F1 expression , 2005, Nature.

[19]  L. Fan,et al.  Germline Genetic Variants Disturbing the Let-7/LIN28 Double-Negative Feedback Loop Alter Breast Cancer Susceptibility , 2011, PLoS genetics.

[20]  Alfredo Hidalgo-Miranda,et al.  Identification and Pathway Analysis of microRNAs with No Previous Involvement in Breast Cancer , 2012, PloS one.

[21]  S. Lowe,et al.  The microcosmos of cancer , 2012, Nature.

[22]  S. Elledge,et al.  Dicer is essential for mouse development , 2003, Nature Genetics.

[23]  A. Gatignol,et al.  The Multiple Functions of TRBP, at the Hub of Cell Responses to Viruses, Stress, and Cancer , 2012, Microbiology and Molecular Reviews.

[24]  R. Shiekhattar,et al.  TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing , 2005, Nature.

[25]  Hyeshik Chang,et al.  Mono-Uridylation of Pre-MicroRNA as a Key Step in the Biogenesis of Group II let-7 MicroRNAs , 2012, Cell.

[26]  Claudia Petritsch,et al.  miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells , 2008 .

[27]  H. Hermeking,et al.  MicroRNAs in the p53 network: micromanagement of tumour suppression , 2012, Nature Reviews Cancer.

[28]  L. Hunyady,et al.  Crosstalk between TGF-β signaling and the microRNA machinery. , 2012, Trends in pharmacological sciences.

[29]  A. Russo,et al.  Dicer and Drosha expression and response to Bevacizumab-based therapy in advanced colorectal cancer patients. , 2013, European journal of cancer.

[30]  A. Goel,et al.  Epigenetics of colorectal cancer. , 2012, Gastroenterology.

[31]  R. Dahiya,et al.  miR-23b represses proto-oncogene Src kinase and functions as methylation-silenced tumor suppressor with diagnostic and prognostic significance in prostate cancer. , 2012, Cancer research.

[32]  Simak Ali,et al.  The DEAD box RNA helicases p68 (Ddx5) and p72 (Ddx17): novel transcriptional co-regulators. , 2008, Biochemical Society transactions.

[33]  Andrea Califano,et al.  The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. , 2010, Cancer cell.

[34]  R. Häsler,et al.  Nuclear death receptor TRAIL-R2 inhibits maturation of let-7 and promotes proliferation of pancreatic and other tumor cells. , 2014, Gastroenterology.

[35]  Sebastian D. Mackowiak,et al.  Circular RNAs are a large class of animal RNAs with regulatory potency , 2013, Nature.

[36]  Paul Graves,et al.  A comprehensive analysis of precursor microRNA cleavage by human Dicer. , 2012, RNA.

[37]  Carla Oliveira,et al.  A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function , 2009, Nature Genetics.

[38]  H. Horvitz,et al.  MicroRNA expression profiles classify human cancers , 2005, Nature.

[39]  E. Izaurralde,et al.  Getting to the Root of miRNA-Mediated Gene Silencing , 2008, Cell.

[40]  F. Slack,et al.  Oncomirs — microRNAs with a role in cancer , 2006, Nature Reviews Cancer.

[41]  J. M. Thomson,et al.  Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing. , 2008, RNA.

[42]  Michael D. Wilson,et al.  ARS2 Is a Conserved Eukaryotic Gene Essential for Early Mammalian Development , 2007, Molecular and Cellular Biology.

[43]  Gregory J. Hannon,et al.  MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies , 2005, Nature Cell Biology.

[44]  S. Cherry,et al.  Ars2 Regulates Both miRNA- and siRNA- Dependent Silencing and Suppresses RNA Virus Infection in Drosophila , 2009, Cell.

[45]  S. Minoshima,et al.  Nucleolar localization of DGCR8 and identification of eleven DGCR8-associated proteins. , 2007, Experimental cell research.

[46]  Hiroshi I. Suzuki,et al.  Modulation of microRNA processing by p53 , 2009, Nature.

[47]  C. Semple,et al.  Posttranscriptional Regulation of miRNAs Harboring Conserved Terminal Loops , 2008, Molecular cell.

[48]  T. Du,et al.  Asymmetry in the Assembly of the RNAi Enzyme Complex , 2003, Cell.

[49]  Gideon Rechavi,et al.  MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth. , 2013, The Journal of clinical investigation.

[50]  Sanjeev Gupta,et al.  Prognostic Significance of Deregulated Dicer Expression in Breast Cancer , 2013, PloS one.

[51]  J. Lovén,et al.  MYCN-regulated microRNAs repress estrogen receptor-α (ESR1) expression and neuronal differentiation in human neuroblastoma , 2010, Proceedings of the National Academy of Sciences.

[52]  Kazuhiro Yoshida,et al.  Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis. , 2010, The Lancet. Oncology.

[53]  Byoung-Tak Zhang,et al.  Molecular Basis for the Recognition of Primary microRNAs by the Drosha-DGCR8 Complex , 2006, Cell.

[54]  Michael T. McManus,et al.  Conditional Loss of Dicer Disrupts Cellular and Tissue Morphogenesis in the Cortex and Hippocampus , 2008, The Journal of Neuroscience.

[55]  M. Esteller,et al.  A precursor microRNA in a cancer cell nucleus , 2011, Cell cycle.

[56]  A. Hata,et al.  Induction of MicroRNA-221 by Platelet-derived Growth Factor Signaling Is Critical for Modulation of Vascular Smooth Muscle Phenotype* , 2009, Journal of Biological Chemistry.

[57]  E. Bandrés,et al.  MicroRNA‐451 Is Involved in the Self‐renewal, Tumorigenicity, and Chemoresistance of Colorectal Cancer Stem Cells , 2011, Stem cells.

[58]  Y. Wan,et al.  Prognostic values of the miR‐17‐92 cluster and its paralogs in colon cancer , 2012, Journal of surgical oncology.

[59]  M. Esteller,et al.  Disrupted microRNA expression caused by Mecp2 loss in a mouse model of Rett syndrome , 2010, Epigenetics.

[60]  Jian-yong Li,et al.  Downregulated Dicer expression predicts poor prognosis in chronic lymphocytic leukemia , 2012, Cancer science.

[61]  C. Kang,et al.  MicroRNA miR-451 downregulates the PI3K/AKT pathway through CAB39 in human glioma , 2011, International journal of oncology.

[62]  M. Fraga,et al.  Genetic unmasking of an epigenetically silenced microRNA in human cancer cells. , 2007, Cancer research.

[63]  S. Diederichs,et al.  Argonaute-3 activates the let-7a passenger strand microRNA , 2013, RNA biology.

[64]  S. Ropero,et al.  A microRNA DNA methylation signature for human cancer metastasis , 2008, Proceedings of the National Academy of Sciences.

[65]  V. Beneš,et al.  Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer , 2012, Oncogene.

[66]  Tae Jin Lee,et al.  p53 regulates epithelial–mesenchymal transition through microRNAs targeting ZEB1 and ZEB2 , 2011, The Journal of experimental medicine.

[67]  D. Matei,et al.  PDGF induced microRNA alterations in cancer cells , 2011, Nucleic acids research.

[68]  Marc Bissonnette,et al.  EGFR Signals Downregulate Tumor Suppressors miR-143 and miR-145 in Western Diet–Promoted Murine Colon Cancer: Role of G1 Regulators , 2011, Molecular Cancer Research.

[69]  Jian Li,et al.  Estrogen Regulates the Tumour Suppressor MiRNA-30c and Its Target Gene, MTA-1, in Endometrial Cancer , 2014, PloS one.

[70]  Milind B. Suraokar,et al.  TAp63 suppresses metastasis through coordinate regulation of Dicer and miRNAs , 2010, Nature.

[71]  A. Pasquinelli,et al.  Auto-regulation of miRNA biogenesis by let-7 and Argonaute , 2012, Nature.

[72]  V. Kim,et al.  The Drosha-DGCR8 complex in primary microRNA processing. , 2004, Genes & development.

[73]  S. Robson,et al.  Human RNA Methyltransferase BCDIN3D Regulates MicroRNA Processing , 2012, Cell.

[74]  J. Yong,et al.  Ars2 Links the Nuclear Cap-Binding Complex to RNA Interference and Cell Proliferation , 2009, Cell.

[75]  Xia Li,et al.  RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators , 2013, Proceedings of the National Academy of Sciences.

[76]  Eugene Berezikov,et al.  microRNAs associated with the different human Argonaute proteins , 2012, Nucleic acids research.

[77]  V. Kim,et al.  The nuclear RNase III Drosha initiates microRNA processing , 2003, Nature.

[78]  C. Croce,et al.  MicroRNAs in Cancer. , 2009, Annual review of medicine.

[79]  R. Shiekhattar,et al.  Human RISC Couples MicroRNA Biogenesis and Posttranscriptional Gene Silencing , 2005, Cell.

[80]  A. Amano,et al.  BRCA1 regulates microRNA biogenesis via the DROSHA microprocessor complex , 2012, The Journal of cell biology.

[81]  M. Esteller,et al.  Dynamic epigenetic regulation of the microRNA-200 family mediates epithelial and mesenchymal transitions in human tumorigenesis , 2011, Oncogene.

[82]  John T. Powers,et al.  Lin28 Enhances Tumorigenesis and is Associated With Advanced Human Malignancies , 2009, Nature Genetics.

[83]  J. Mendell,et al.  Myc: Maestro of MicroRNAs. , 2010, Genes & cancer.

[84]  S. Jayasena,et al.  Functional siRNAs and miRNAs Exhibit Strand Bias , 2003, Cell.

[85]  David L. A. Wood,et al.  MicroRNAs and their isomiRs function cooperatively to target common biological pathways , 2011, Genome Biology.

[86]  F. Fuller-Pace.  DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation , 2006, Nucleic acids research.

[87]  Shuang Huang,et al.  Signaling by p38 MAPK Stimulates Nuclear Localization of the Microprocessor Component p68 for Processing of Selected Primary MicroRNAs , 2013, Science Signaling.

[88]  H. Kalofonos,et al.  Prognostic Significance of Dicer Cellular Levels in Soft Tissue Sarcomas , 2012, Cancer investigation.

[89]  Yan Wang,et al.  EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2 , 2013, Nature.

[90]  Etienne Gagnon,et al.  Alternative RISC assembly: binding and repression of microRNA-mRNA duplexes by human Ago proteins. , 2012, RNA.

[91]  M. Siomi,et al.  Posttranscriptional regulation of microRNA biogenesis in animals. , 2010, Molecular cell.

[92]  G. Tell,et al.  Expression of Dicer and Drosha in triple-negative breast cancer , 2012, Journal of Clinical Pathology.

[93]  Martin J. Simard,et al.  Argonaute proteins: key players in RNA silencing , 2008, Nature Reviews Molecular Cell Biology.

[94]  M. Esteller,et al.  CpG island hypermethylation-associated silencing of non-coding RNAs transcribed from ultraconserved regions in human cancer , 2010, Oncogene.

[95]  Sung-Chou Li,et al.  Silencing of miR-1-1 and miR-133a-2 cluster expression by DNA hypermethylation in colorectal cancer. , 2012, Oncology reports.

[96]  S. Diederichs,et al.  Loop-miRs: active microRNAs generated from single-stranded loop regions , 2013, Nucleic acids research.

[97]  E. Furth,et al.  Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster , 2006, Nature Genetics.

[98]  Kevin J Luebke,et al.  Faculty Opinions recommendation of The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. , 2009 .

[99]  A. Hata,et al.  Smad proteins bind a conserved RNA sequence to promote microRNA maturation by Drosha. , 2010, Molecular cell.

[100]  L. Paz-Ares,et al.  DICER1, DROSHA and miRNAs in patients with non-small cell lung cancer: implications for outcomes and histologic classification. , 2013, Carcinogenesis.

[101]  Hanah Margalit,et al.  Clustering and conservation patterns of human microRNAs , 2005, Nucleic acids research.

[102]  C. Joo,et al.  TUT4 in Concert with Lin28 Suppresses MicroRNA Biogenesis through Pre-MicroRNA Uridylation , 2009, Cell.

[103]  C. Xue,et al.  Dynamic Localisation of Mature MicroRNAs in Human Nucleoli is Influenced by Exogenous Genetic Materials , 2013, PloS one.

[104]  Gretchen M. Williams,et al.  DICER1 Mutations in Familial Pleuropulmonary Blastoma , 2009, Science.

[105]  Michael A. Beer,et al.  Lin-28B transactivation is necessary for Myc-mediated let-7 repression and proliferation , 2009, Proceedings of the National Academy of Sciences.

[106]  D. Haussler,et al.  Posttranscriptional Crossregulation between Drosha and DGCR8 , 2009, Cell.

[107]  C. Deng,et al.  Tumor suppressor BRCA1 epigenetically controls oncogenic microRNA-155 , 2011, Nature Medicine.

[108]  J. Norman,et al.  Mutant p53 Regulates Dicer through p63-dependent and -independent Mechanisms to Promote an Invasive Phenotype* , 2013, The Journal of Biological Chemistry.

[109]  Arndt Borkhardt,et al.  MicroRNAs Distinguish Cytogenetic Subgroups in Pediatric AML and Contribute to Complex Regulatory Networks in AML-Relevant Pathways , 2013, PloS one.

[110]  B. Cullen,et al.  Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. , 2004, RNA.

[111]  E. Makeyev,et al.  Argonaute identity defines the length of mature mammalian microRNAs , 2012, Nucleic acids research.

[112]  C. Croce,et al.  A microRNA expression signature of human solid tumors defines cancer gene targets , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[113]  D. Cantú de León,et al.  Disruption of CTCF at the miR-125b1 locus in gynecological cancers , 2012, BMC Cancer.

[114]  S. Kakar,et al.  MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer. , 2011, Carcinogenesis.

[115]  Shuta Tomida,et al.  Reduced expression of Dicer associated with poor prognosis in lung cancer patients , 2005, Cancer science.

[116]  D. Court,et al.  Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage. , 2001, Structure.

[117]  J. Kjems,et al.  Natural RNA circles function as efficient microRNA sponges , 2013, Nature.

[118]  Shuomin Zhu,et al.  MicroRNA-21 Targets the Tumor Suppressor Gene Tropomyosin 1 (TPM1)* , 2007, Journal of Biological Chemistry.

[119]  Tsung-Cheng Chang,et al.  Widespread microRNA repression by Myc contributes to tumorigenesis , 2008, Nature Genetics.

[120]  R. Siebert,et al.  Epigenetic silencing of the tumor suppressor microRNA Hsa-miR-124a regulates CDK6 expression and confers a poor prognosis in acute lymphoblastic leukemia. , 2009, Cancer research.

[121]  E. Lai,et al.  Functional parameters of Dicer-independent microRNA biogenesis. , 2012, RNA.

[122]  B. Cullen,et al.  Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. , 2003, Genes & development.

[123]  Jing Wang,et al.  Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes , 2008, Nature Immunology.

[124]  H. Ishii,et al.  Dicer 1, ribonuclease type III modulates a reprogramming effect in colorectal cancer cells. , 2012, International journal of molecular medicine.

[125]  Frank Speleman,et al.  miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis , 2010, Nature Cell Biology.

[126]  P. Gunaratne,et al.  Modulation of microRNA Activity by Semi-microRNAs , 2012, Front. Gene..

[127]  L. Gao,et al.  MicroRNA-193a represses c-kit expression and functions as a methylation-silenced tumor suppressor in acute myeloid leukemia , 2011, Oncogene.

[128]  I. Bozzoni,et al.  Primary microRNA transcripts are processed co-transcriptionally , 2008, Nature Structural &Molecular Biology.

[129]  Yukio Kawahara,et al.  RNA editing of the microRNA‐151 precursor blocks cleavage by the Dicer–TRBP complex , 2007, EMBO reports.

[130]  L. Lim,et al.  Transcripts Targeted by the MicroRNA-16 Family Cooperatively Regulate Cell Cycle Progression , 2007, Molecular and Cellular Biology.

[131]  W. Gerald,et al.  Endogenous human microRNAs that suppress breast cancer metastasis , 2008, Nature.

[132]  Domenico Coppola,et al.  MicroRNA-155 Is Regulated by the Transforming Growth Factor β/Smad Pathway and Contributes to Epithelial Cell Plasticity by Targeting RhoA , 2008, Molecular and Cellular Biology.

[133]  Jan-Fang Cheng,et al.  Dicer, Drosha, and outcomes in patients with ovarian cancer. , 2008, The New England journal of medicine.

[134]  Zhuling Yang,et al.  Immunohistochemical study of Dicer and Drosha expression in the benign and malignant lesions of gallbladder and their clinicopathological significances. , 2012, Pathology, research and practice.

[135]  Eugene Berezikov,et al.  Mammalian mirtron genes. , 2007, Molecular cell.

[136]  A. Hata,et al.  Regulation of MicroRNA Biogenesis: A miRiad of mechanisms , 2009, Cell Communication and Signaling.

[137]  V. Kim,et al.  The role of PACT in the RNA silencing pathway , 2006, The EMBO journal.

[138]  L. Lim,et al.  A microRNA component of the p53 tumour suppressor network , 2007, Nature.

[139]  E. Schuman,et al.  Deep Sequencing and High-Resolution Imaging Reveal Compartment-Specific Localization of Bdnf mRNA in Hippocampal Neurons , 2013, Science Signaling.

[140]  P. Rennie,et al.  Androgen-regulated processing of the oncomir miR-27a, which targets Prohibitin in prostate cancer. , 2012, Human Molecular Genetics.

[141]  Sven Diederichs,et al.  Argonaute proteins regulate microRNA stability: Increased microRNA abundance by Argonaute proteins is due to microRNA stabilization , 2011, RNA biology.

[142]  B. Cullen,et al.  Sequence requirements for micro RNA processing and function in human cells. , 2003, RNA.

[143]  K. Claffey,et al.  Argonaute-2 expression is regulated by epidermal growth factor receptor and mitogen-activated protein kinase signaling and correlates with a transformed phenotype in breast cancer cells. , 2009, Endocrinology.

[144]  Sanghyuk Lee,et al.  MicroRNA genes are transcribed by RNA polymerase II , 2004, The EMBO journal.

[145]  B. Cullen,et al.  Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. , 2004, Nucleic acids research.

[146]  Sam Griffiths-Jones,et al.  MicroRNAs from the same precursor have different targeting properties , 2012, Silence.

[147]  Hiroyuki Yamamoto,et al.  A genetic defect in exportin-5 traps precursor microRNAs in the nucleus of cancer cells. , 2010, Cancer cell.

[148]  C. Croce,et al.  p53 regulates epithelial–mesenchymal transition through microRNAs targeting ZEB1 and ZEB2 , 2011, The Journal of experimental medicine.

[149]  Min Li,et al.  Effect of miR-451 on the Biological Behavior of the Esophageal Carcinoma Cell Line EC9706 , 2012, Digestive Diseases and Sciences.

[150]  M. Martinka,et al.  Expression of the RNase III enzyme DROSHA is reduced during progression of human cutaneous melanoma , 2013, Modern Pathology.

[151]  Kai-Fai Lee,et al.  MicroRNA-34a suppresses invasion through downregulation of Notch1 and Jagged1 in cervical carcinoma and choriocarcinoma cells. , 2010, Carcinogenesis.

[152]  J. Ule,et al.  Selective inhibition of microRNA accessibility by RBM38 is required for p53 activity , 2011, Nature communications.

[153]  Yang Shi,et al.  Hypoxia Potentiates MicroRNA-Mediated Gene Silencing through Posttranslational Modification of Argonaute2 , 2011, Molecular and Cellular Biology.