**3. Alteration of miRNA biogenesis in response to DNA damage and repair**

Because miRNAs are actively involved in regulation of genes that are related to DNA damage and repair, it was not surprising to find that miRNA biogenesis changes in response to DNA damage and repair. Several studies demonstrated that both miRNA transcription and maturation process are altered in response to DNA damage and repair.

Recent studies show that transcription of miRNA can be directly affected by DNA damage. The *P53* gene plays a critical role in this regulation. For example, miR-34a can be up-regulated by the *P53* gene in response to DNA damage (Chang et al., 2007; Corney et al., 2007; He et al., 2007; Raver-Shapira et al., 2007; Welch et al., 2007). Up-regulation of miR-34a results in apoptosis, cell-cycle arrest, and DNA repair. miR-34a is a direct transcriptional target of P53 because the promoter region of miR-34a contains a canonical P53 binding site. When DNA damage activates the *P53* gene, P53 protein binds to the promoter of miR-34a and up-regulates miRNA expression. In *Caenorhabditis elegans*, miR-34a expression was enhanced by irradiation in a P53 independent manner, and knocking down of the *Cep1* gene (homolog of the *P53* gene) had no effect on the miR-34a response to irradiation (Kato et al., 2009). Up-regulation of miR-34a in response to genotoxin exposure is also observed in different biological systems (Chen et al., 2011; Li et al., 2010;

original sequence using the sister chromatid as a template. NHEJ is a relatively simple way for DNA double-strand repair and it just rejoins two broken ends without correcting any

A microRNA gene can be located in an intron of another gene, in either the sense or antisense orientation. miRNA can be coordinately expressed with its host gene, or it can have its own promoter independent of its host gene (Ozsolak et al., 2008). The biogenesis of miRNA is a complex process as shown in Figure 1. miRNA is first transcribed as a long primary miRNA (pri-miRNA) by RNA polymerase II in the nucleus (Lee et al., 2004). PrimiRNA is structurally similar to mRNA, but contains a stable stem-loop structure (Cai et al., 2004). Recognition of the hairpin and selection of a cleavage site are mediated by DGCR8. Nuclear RNase III (Drosha) then cleaves the pri-miRNA to release the hairpin-shaped precursor miRNA (pre-miRNAs). The pre-miRNA is exported from the nucleus to the cytoplasm by Exportin 5 (Exp5). In the cytoplasm, the pre-miRNA is subsequently cut by cytoplasmic RNase III (Dicer) in complex with Argonaute2 (Ago2) and TRBP, a doublestranded RNA-binding protein. This process cleaves the pre-miRNA hairpins to remove its hairpin loop, resulting in a miRNA duplex with the appropriate length (Gregory et al., 2005; Han et al., 2004; Lee et al., 2003). Normally, one strand of the duplex is then degraded. The mature miRNA are incorporated into an RNA-induced silencing complex (RISC) (Gregory, et al., 2005; Grishok et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001; Maniataki and Mourelatos, 2005). RISC recognizes target mRNAs through full or partial base-pairing interactions between the miRNA and the to "3'-untranslated region (UTR) of the target mRNA. Depending on pairing interactions between miRNAs and their targets, miRNAs suppress their target gene expression by either mRNA cleavage or translational repression. If an mRNA target match perfectly or near-perfectly to the miRNA, the mRNA will be degraded;

otherwise, the mRNA will be translationally suppressed (Meister and Tuschl, 2004).

and maturation process are altered in response to DNA damage and repair.

**3. Alteration of miRNA biogenesis in response to DNA damage and repair** 

Because miRNAs are actively involved in regulation of genes that are related to DNA damage and repair, it was not surprising to find that miRNA biogenesis changes in response to DNA damage and repair. Several studies demonstrated that both miRNA transcription

Recent studies show that transcription of miRNA can be directly affected by DNA damage. The *P53* gene plays a critical role in this regulation. For example, miR-34a can be up-regulated by the *P53* gene in response to DNA damage (Chang et al., 2007; Corney et al., 2007; He et al., 2007; Raver-Shapira et al., 2007; Welch et al., 2007). Up-regulation of miR-34a results in apoptosis, cell-cycle arrest, and DNA repair. miR-34a is a direct transcriptional target of P53 because the promoter region of miR-34a contains a canonical P53 binding site. When DNA damage activates the *P53* gene, P53 protein binds to the promoter of miR-34a and up-regulates miRNA expression. In *Caenorhabditis elegans*, miR-34a expression was enhanced by irradiation in a P53 independent manner, and knocking down of the *Cep1* gene (homolog of the *P53* gene) had no effect on the miR-34a response to irradiation (Kato et al., 2009). Up-regulation of miR-34a in response to genotoxin exposure is also observed in different biological systems (Chen et al., 2011; Li et al., 2010;

deletions or rearrangements of DNA.

**2. Biogenesis of miRNA** 

Li et al., 2011; Zenz et al., 2009). miR-34c, another member of miR-34 family, is transcriptionally up-regulated by P53 following DNA damage (Cannell et al., 2010). In addition to miR-34a, P53 can also regulate the expression of miR-192, miR-194, and miR-215. These miRNAs are considered tumor suppressor miRNAs (Braun et al., 2008; Georges et al., 2008).

miRNA biogenesis is globally induced upon DNA damage in an ATM (ataxia telangiectasia mutated) dependent manner (Zhang et al., 2011). The ATM gene encodes a DNA damageinducible kinase. ATM controls cell grow rate by interacting with other proteins, for example BRCA1, following DNA damage. In response to strand breaks or other type of DNA damage, the ATM protein coordinates DNA repair by activating other proteins. Because of its central role in cell division and DNA repair, the ATM protein is important in carcinogenesis. More than one-fourth of miRNAs were significantly upregulated after DNA damage, while loss of ATM activity abolished their induction. Their results show that DNA damage activates the ATM kinase that directly binds to and phosphorylates KH-type splicing regulatory protein (KSRP), leading to enhanced interaction between KSRP and primiRNAs and increased KSRP activity in miRNA processing. The increased activity, in turn, results in more pre-miRNAs from pri-miRNAs, so that more miRNA products are produced to respond to the DNA damage.

Other studies show a different mechanism by which DNA damage signaling is linked to the miRNA maturation processes. Several miRNAs with growth suppressive function, including miR-16-1, miR-143 and miR-145, were regulated at the post transcriptional level through a P53-mediated miRNA maturation process in response to DNA damage (Suzuki et al., 2009; Toledo and Bardot, 2009). The P53 tumor suppressor protein binds to Drosha to facilitate the processing of pri-miRNAs to pre-miRNAs. Mutation in the DNA-binding domain of P53 decreases processing of pri-miRNAs by Drosha, and reduces the expression of the related miRNAs. In silico analyses, all three component of the P53 tumor suppressor, P53, P63, and P73, can regulate the major components of miRNA processing, such as Drosha-DGCR8, Dicer-TRBP2, and Agronaute proteins. Thus, when DNA damage activates the P53 gene, the activated P53 gene can modulate miRNA expression by affecting the miRNA biogenesis processes.

miR-24 regulates the DNA damage response by down-regulation of H2AX, the initial sensor protein for the DNA damage response. miR-100, miR-101 and miR-421 suppress ATM, the chief transducer of the DNA damage response, by targeting the 3'-UTR of ATM. miR-16 can up-regulate ATM activity by suppressing levels of Wip1. DNA repair pathways are regulated by a number of miRNAs involved in different types of DNA damage correction. the NER protein RAD23B was down-regulated by miR-373. MMR protein MSH2 and MSH6 were down regulated by miR-21 and MLH1/MSH2 were suppressed by miR-155. The HRR protein BRCA1 was down-regulated by miR-182 and RAD52 was suppressed by miR-210 and miR-373. The NHEJ protein DNA-PKcs was suppressed by miR-101 (Yan, Ng. 2010).
