The role of ATM in the cell cycle control of V(D)J recombination

Lymphocyte development requires the assembly of diversified antigen receptor complexes generated by the genetically programmed V(D)J recombination event. Because germline DNA is cut, introducing potentially dangerous double-stranded breaks (DSBs) and rearranged prior to repair, its activity is limited to the non-cycling stages of the cell cycle, G0/G1. The potential involvement of a key mediator, Ataxia Telangiectasia Mutated or ATM, in the DNA damage response (DDR) and cell cycle checkpoints has been implicated in recombination, but its role is not fully understood. Thymic lymphomas from ATM deficient mice contain clonal chromosomal translocations involving the T-cell antigen receptor (TCR). A previous report found ATM and its downstream target p53 associated with V(D)J intermediates, suggesting the DDR senses recombination. In this study, we sought to understand the role of ATM in V(D)J recombination. Developing thymocytes from ATM deficient mice were analyzed according to the cell cycle to detect V(D)J intermediates. Examination of all TCR loci in the non-cycling (G0/G1) and cycling (S/G2/M) fractions revealed the persistence of intermediates in ATM deficient thymocytes, contrary to the wild-type in which intermediates are found only during G0/G1. Further analysis found no defect in end-joining of intermediates, nor were they detected in developed T-cells. Based upon the presence of persisting intermediates, the recombination initiating nuclease Rag-2 was examined; strict regulation limits it to G 0/G1. Rag-2 regulation was not affected by an ATM deficiency as Rag-2 expression remained contained within G0/G 1, indicating recombination is not continuous. To determine if an ATM deficiency affects recognition of V(D)J breaks, sites of recombination identified by a TCR locus or Rag expression were analyzed according to co-localization with a DDR factor phosphorylated immediately after DNA damage, phosphorylated H2AX (γH2AX). No differences in co-localization were found between the wild-type and ATM deficiency, demonstrating ATM deficient lymphocytes retain the ability to recognize DSBs. Together, these results suggest ATM is necessary in the cell cycle regulation of recombination but not essential for the identification of V(D)J breaks. ATM ensures the containment of intermediates within G0/G1 and maintains genomic stability of developing lymphocytes, emphasizing its fundamental role in preventing tumorigenesis.^

Stability and potential application of spliced nuclear pre -mRNA introns

A major portion of this thesis work was dedicated to study the nature and significance of spliced introns. The initial work was focused on studying the IVS1$\sb{\rm C\beta 1}$ intron from a T-cell receptor (TCR)-$\beta$ gene. Compared to an intron lariat control from adenovirus pre-mRNA that was spliced in vitro, IVS1$\sb{\rm C\beta 1}$ was debranched less efficiently by HeLa S100 extracts, although IVS1$\sb{\rm C\beta 1}$ also used the consensus branchpoint in vivo. Subcellular-fractionation analysis showed that most IVS1$\sb{\rm C\beta 1}$ lariats cofractionated with pre-mRNA in the nucleus, consistent with the possibility that intron degradation releases splicing factors which will be available for further rounds of splicing. The half-life of IVS1$\sb{\rm C\beta 1}$ from the endogenous TCR-$\beta$ gene was measured using the general transcription inhibitor actinomycin D to be about $\sim$15 min, which was similar to that of unstable mRNAs such as c-myc mRNA.^ The general transcription inhibitor DRB was also used for intron stability analysis. Unexpectedly, DRB decreased intron and pre-mRNA levels only initially, it later increased the levels of intron-containing RNAs. Inhibition of transcription initiation appeared to be the major early effect (the reduction phase); whereas enhanced premature transcription termination was dominant later (the induction phase).^ Having established the procedures for studying in vivo spliced introns, this approach was applied to study the mechanism of nonsense-mediated downregulation (NMD), a phenomena in which premature termination codons (PTCs) decrease the levels of mRNAs. In this study, the novel intron-oriented approach was applied to study the mechanism of NMD. The levels of spliced introns immediately upstream and downstream of a PTC-bearing exon in a TCR-$\beta$ gene were identified and analyzed along with their pre-mRNA. Although PTC reduced the mRNA levels by 4 to 9 fold, the steady-state levels of spliced introns and the pre-mRNA-to-intron ratios were not significantly altered, indicating that the PTC did not significantly inhibit TCR-$\beta$ RNA splicing. Consistent with this conclusion, the half-lives of the PTC$\sp+$ and PTC$\sp-$ pre-mRNA were similar. The protein synthesis inhibitor cyclohexmide (CHX) upregulated the levels of the PTC$\sp+$ mRNA over 10 fold without affecting the levels of the spliced introns, suggesting that the reversal effect of CHX was through stabilization, not production. These results indicated that inhibition of splicing could not be the major mechanism for the NMD pathway of the TCR-$\beta$ gene, instead, suggesting that mRNA destabilization may be more important. (Abstract shortened by UMI.) ^

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^