The Fanconi anemia pathway is required for the efficient repair of damaged DNA. A key step in this pathway is the monoubiquitination of the FANCD2 protein by the ubiquitin ligase (E3) composed of Fanconi anemia core complex proteins. Here, we show that UBE2T is the ubiquitin-conjugating enzyme (E2) essential for this pathway. UBE2T binds to FANCL, the ubiquitin ligase subunit of the Fanconi anemia core complex, and is required for the monoubiquitination of FANCD2 in vivo. DNA damage in UBE2T-depleted cells leads to the formation of abnormal chromosomes that are a hallmark of Fanconi anemia. In addition, we show that UBE2T undergoes automonoubiquitination in vivo. This monoubiquitination is stimulated by the presence of the FANCL protein and inactivates UBE2T. Therefore, UBE2T is the E2 in the Fanconi anemia pathway and has a self-inactivation mechanism that could be important for negative regulation of the Fanconi anemia pathway.

MicroRNAs (miRNAs) are key regulators of gene expression. They are conserved across species, expressed across cell types, and active against a large proportion of the transcriptome. The sequence-complementary mechanism of miRNA activity exploits combinatorial diversity, a property conducive to network-wide regulation of gene expression, and functional evidence supporting this hypothesized systems-level role has steadily begun to accumulate. The emerging models are exciting and will yield deep insight into the regulatory architecture of biology. However, because of the technical challenges facing the network-based study of miRNAs, many gaps remain. Here, we review mammalian miRNAs by describing recent advances in understanding their molecular activity and network-wide function. Keywords microRNA; miRNA; biogenesis; Drosha; DGCR8; Dicer; TRBP; gene expression; network; system MicroRNAs (miRNAs) are ~22-nucleotide RNAs that post-transcriptionally repress gene expression by base-pairing to mRNAs. More than half of all mRNAs are estimated to be targets of miRNAs and each miRNA is predicted to regulate up to hundreds of targets. Consistent with this pervasive activity, miRNAs regulate a broad range of processes, including proliferation, differentiation, and apoptosis. These short RNAs are particularly critical during development, their total loss in the embryo leading to lethality. Although many studies focus on binary miRNA-target interactions in defining phenotypes, it is becoming increasingly evident that each miRNA exerts its influence by targeting multiple functionally-related genes that constitute gene expression networks. In this review, we provide a network-level perspective of mammalian miRNAs and describe their genomic organization, molecular activity, and biological function. The molecular biology of miRNAs Biogenesis and genomic organizationThe biogenesis of miRNAs has been reviewed extensively elsewhere 1 and is summarized briefly here. Genes encoding miRNAs are transcribed by RNA Polymerase II into long primary transcripts (pri-miRNAs) that are processed by the RNase III enzyme Drosha to © 2013 Elsevier Ltd. All rights reserved. ‡ To whom correspondence should be addressed. gurtan@mit.edu or sharppa@mit.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Figure 1). Pre-miRNAs are subsequently transported into the nucleus by Exportin 5 3; 4; 5 and then processed by another RNase III enzyme, Dicer, to yield a mature miRNA as well as a star strand that is degraded 6; 7; 8; 9; 10 . The miRNA is then loaded into an Argonaute protein within the RNA induced sil...

IntroductionThe 13 identified Fanconi anemia (FA) proteins cooperate in a common cellular pathway regulating the cellular response to DNA cross-linking agents, such as cisplatin (CDDP), diepoxybutane (DEB), and mitomycin C (MMC). 1 Of these FA proteins, 8 (A, B, C, E, F, G, L, and M) are assembled into a core complex, 2,3 which contains a ubiquitin E3 ligase activity (FANCL subunit) 4 and a DNA translocase activity (FANCM). 5 In response to DNA damage, or during S-phase progression, the FA core complex coordinately monoubiquitinates 2 downstream substrates, FANCD2 6,7 and FANCI. [8][9][10] These monoubiquitinated proteins subsequently translocate to the chromatin where they are believed to interact with additional downstream FA proteins, including FANCJ/BRIP1, [11][12][13] FANCD1/BRCA2, 14 and FANCN/PALB2. 15,16 These downstream proteins then regulate homologous recombination (HR) repair. Disruption of any of the proteins in the FA pathway accounts for the common cellular and clinical phenotype of FA patients. 17 How the pathway participates in the process of DNA cross-link repair remains unknown. 18 Some FA complementation groups also exhibit additional phenotypic variation, suggesting that some FA genes have functions outside a simple linear FA pathway. [19][20][21][22] The FA core complex may have additional functions beyond the monoubiquitination of FANCD2 and FANCI. 8 A FANCD2-Ub linear fusion protein complements the MMC hypersensitivity of Fancd2-deficient chicken cells, but fails to correct the phenotype of FA core complex-deficient cells. 23 The FA core complex may therefore have additional activities, such as the recognition of specific DNA substrates, the regulation of the DNA replication machinery, and/or the monoubiquitination of additional (unknown) substrates. These additional functions may be explained, at least in part, by the presence of FA core subcomplexes with variable sizes and variable subcellular distributions. 3 When a replication fork encounters an interstrand DNA crosslink during replication, the replication fork arrests near the lesion, resulting in aberrant DNA structures. These abnormal structures activate checkpoint and repair pathways. FA cells, carrying mutations in FA genes, are highly sensitive to DNA crosslinking agents, compared with other DNA-damaging agents, such as ionizing radiation (IR), ultraviolet (UV), and hydroxyurea (HU). This hypersensitivity suggests that some components of the FA core complex may be involved in detecting and binding the DNA lesions caused by treatment of DNA crosslinking agents.The recently identified FANCM 5 and FANCJ 11-13 proteins suggest a direct involvement of FA proteins at sites of DNA repair. FANCM is homologous to the archaeal protein Hef (helicaseassociated endonuclease for fork-structured DNA), and is a member of the XP-F superfamily. 24 The N-terminal region of FANCM is able to bind to single-stranded DNA. 25 Moreover, FANCM has a DNA-dependent ATPase activity, and it can dissociate DNA triple helices in vitro. 5,25 FANCJ/BRIP1, whi...