Evidence that a mutation in the MLH1 3'-untranslated region confers a mutator phenotype and mismatch repair deficiency in patients with relapsed leukemia

Defects in DNA mismatch repair (MMR) are the molecular basis of certain cancers, including hematological malignancies. The defects are often caused by mutations in coding regions of MMR genes or promoter methylation of the genes. However, in many cases, despite that a hypermutable phenotype is detected in a patient, no mutations/hypermethylations of MMR genes can be detected. We report here a novel mechanism that a mutation in the MLH1 3'-untranslated region (3'-UTR) leads to MMR deficiency. A relapsed leukemia patient displayed microsatellite instability, but no genetic and epigenetic alterations in key MMR genes were identifiable. Instead, a 3-nucleotide (TTC) deletion in the MLH1 3'-UTR was found in the patient's blood sample. The mutant MLH1 3'-UTR was found to significantly reduce the expressions of both a firefly luciferase reporter gene and an ectopic MLH1 gene in model cell lines. Consistent with these observations, a significant reduction in the steady-state level of MLH1 mRNA was observed in white blood cells of the patient. These findings suggest that the mutant MLH1 3'-UTR can cause a severely reduced/defective MMR activity conferring leukemia relapse, likely by down-regulating MLH1 expression at the mRNA level. Although the exact mechanism by which the mutant 3'-UTR down-regulates the MLH1 mRNA is not known, our findings provide a novel marker for cancers with MMR defects.     

The relationship between microsatellite slippage mutation rate and the number of repeat units

Microsatellite markers are widely used for genetic studies, but the relationship between microsatellite slippage mutation rate and the number of repeat units remains unclear. In this study, microsatellite distributions in the human genome are collected from public sequence databases. We observe that there is a threshold size for slippage mutations. We consider a model of microsatellite mutation consisting of point mutations and single stepwise slippage mutations. From two sets of equations based on two stochastic processes and equilibrium assumptions, we estimate microsatellite slippage mutation rates without assuming any relationship between microsatellite slippage mutation rate and the number of repeat units. We use the least squares method with constraints to estimate expansion and contraction mutation rates. The estimated slippage mutation rate increases exponentially as the number of repeat units increases. When slippage mutations happen, expansion occurs more frequently for short microsatellites and contraction occurs more frequently for long microsatellites. Our results agree with the length-dependent mutation pattern observed from experimental data, and they explain the scarcity of long microsatellites.     

Somatic frameshift mutations in the Bloom syndrome BLM gene are frequent in sporadic gastric carcinomas with microsatellite mutator phenotype

Background  

Genomic instability has been reported at microsatellite tracts in few coding sequences. We have shown that the Bloom syndrome BLM gene may be a target of microsatelliteinstability (MSI) in a short poly-adenine repeat located in its coding region. To further characterize the involvement of BLM in tumorigenesis, we have investigated mutations in nine genes containing coding microsatellites in microsatellite mutator phenotype (MMP) positive and negative gastric carcinomas (GCs).     

Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times, which cause their genome-wide underrepresentation

Microsatellites are short tandemly repeated DNA sequence motifs that are highly variable in most organisms. In contrast to mammals, long microsatellites (>15 repeats) are extremely rare in the Drosophila melanogaster genome. To investigate this paucity of long microsatellites in Drosophila, we studied 19 loci with exceptionally long microsatellite alleles. Inter- and intraspecific analysis showed that long microsatellite alleles arose in D. melanogaster only very recently. This lack of old alleles with many repeats indicated that long microsatellite alleles have short persistence times. The size distribution of microsatellite mutations in mutation-accumulation lines suggests that long alleles have a mutation bias toward a reduction in the number of repeat units. This bias causes the short persistence times of long microsatellite alleles. We propose that species-specific, size-dependent mutation spectra of microsatellite alleles may provide a general mechanism to account for the observed differences in microsatellite length between species.     

Separate cis-trans pathways post-transcriptionally regulate murine CD154 (CD40 ligand) expression: a novel function for CA repeats in the 3'-untranslated region

We report a role for CA repeats in the 3'-untranslated region (3'-UTR) in regulating CD154 expression. Human CD154 is encoded by an unstable mRNA; this instability is conferred in cis by a portion of its 3'-UTR that includes a polypyrimidine-rich region and CA dinucleotide repeat. We demonstrate similar instability activity with the murine CD154 3'-UTR. This instability element mapped solely to a conserved 100-base CU-rich region alone, which we call a CU-rich response element. Surprisingly, the CA dinucleotide-rich region also regulated reporter expression but at the level of translation. This activity was associated with poly(A) tail shortening and regulated by heterogeneous nuclear ribonucleoprotein L levels. We conclude that the CD154 3'-UTR contains dual cis-acting elements, one of which defines a novel function for exonic CA dinucleotide repeats. These findings suggest a mechanism for the association of 3'-UTR CA-rich response element polymorphisms with CD154 overexpression and the subsequent risk of autoimmune disease.     

大蹄蝠全基因组微卫星分布特征分析

脊椎动物基因组含有丰富的微卫星信息。本研究对翼手目动物中的大蹄蝠全基因组及其基因的微卫星分布特征进行分析,并对含有微卫星编码序列的基因进行注释分析。结果表明,大蹄蝠全基因组大小为2.24 Gb,共含有497 883个微卫星,其中,数量和比例最多的是单碱基和二碱基重复类型,分别有173 953个(34.94%)和222 591个(44.71%),相对丰度分别为77.78 loci/Mb和99.52 loci/Mb。微卫星数量从单碱基重复到六碱基重复单元最多的类型分别为(A)n、(AC)n、(TAT)n、(TTTA)n、(AACAA)n和(TATCTA)n,比例分别为95.14%、55.25%、38.41%、22.17%、48.68%和20.30%。不同基因区和基因间区的数量及丰度不同,其中基因间区的微卫星数量及其丰度最大,分别为322 666个和2 541.57 loci/Mb,编码区的微卫星数量及其丰度最小,分别为1 461个和461.98 loci/Mb。基因间区和全基因组的微卫星的分布特征相似。编码区最多的微卫星类型为三碱基重复单元,外显子最多的微卫星类型为单碱基、二碱基和三碱基重...     

Low abundance of Escherichia coli microsatellites is associated with an extremely low mutation rate

It is widely assumed that microsatellites are generated by replication slippage, a mutation process specific to repetitive DNA. Consistent with their high mutation rate, microsatellites are highly abundant in most eukaryotic genomes. In Escherichia coli, however, microsatellites are rare and short despite the fact that a high microsatellite mutation rate was described. We show that this high microsatellite instability depends on the presence of the F-plasmid. E. coli cells lacking the F-plasmid have extremely low microsatellite mutation rates. This result provides a possible explanation for the genome-wide low density of microsatellites in E. coli. Furthermore, we show that the F-plasmid induced microsatellite instability is independent of the mismatch repair pathway.     

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.      

Quantifying ascertainment bias and species-specific length differences in human and chimpanzee microsatellites using genome sequences

Surveys of variability of homologous microsatellite loci among species reveal an ascertainment bias for microsatellite length where microsatellite loci isolated in one species tend to be longer than homologous loci in related species. Here, we take advantage of the availability of aligned human and chimpanzee genome sequences to compare length difference of homologous microsatellites for loci identified in humans to length difference for loci identified in chimpanzees. We are able to quantify ascertainment bias for a range of motifs and microsatellite lengths. Because ascertainment bias should not exist if a microsatellite selected in one species is as likely to be longer as it is to be shorter than its homologue, we propose that the nature of ascertainment bias can provide evidence for understanding how microsatellites evolve. We show that bias is greater for longer microsatellites but also that many long microsatellites have short homologues. These results are consistent with the notion that growth of long microsatellites is constrained by an upper length boundary that, when reached, sometimes results in large deletions. By evaluating ascertainment bias separately for interrupted and uninterrupted repeats we also show that long microsatellites tend to become interrupted, thereby contributing a second component of ascertainment bias. Having accounted for ascertainment bias, in agreement with results published elsewhere, we find that microsatellites in humans are longer on average than those in chimpanzees. This length difference is similar among repeat motifs but surprisingly comprises two roughly equal components, one associated with the repeats themselves and one with the flanking sequences. The differences we find can only be explained if microsatellites are both evolving directionally under a biased mutation process and are doing so at different rates in different closely related species.     

The proximal region of the 3'-untranslated region of cyclooxygenase-2 is recognized by a multimeric protein complex containing HuR,TIA-1, TIAR,and the heterogeneous nuclear ribonucleoprotein U

Cyclooxygenase-2 (COX-2) is an early response gene induced in renal mesangial cells by interleukin-1beta (IL-1beta). The 3'-untranslated region (3'-UTR) of COX-2 mRNA plays an important role in IL-1beta induction by regulating message stability and translational efficiency. The first 60 nucleotides of the 3'-UTR of COX-2 are highly conserved and contain multiple copies of the regulatory sequence AUUUA. Introduction of the 60-nucleotide sequence into the 3'-UTR of a heterologous reporter gene resulted in a 70% decrease in reporter gene expression. Electrophoretic mobility shift assays (EMSAs) demonstrated that mesangial cell nuclear fractions contain a multimeric protein complex that bound this region of COX-2 mRNA in a sequence-specific manner. We identified four members of the protein-RNA complex as HuR, TIA-1, TIAR, and the heterogeneous nuclear ribonucleoprotein U (hnRNP U). Treatment of mesangial cells with IL-1beta caused an increase in cytosolic HuR, which was accompanied by an increase in COX-2 mRNA that co-immunoprecipitated with cytosolic HuR. Therefore, we propose that HuR binds to the proximal region of the 3'-UTR of COX-2 following stimulation by IL-1beta and increases the expression of COX-2 mRNA by facilitating its transport out of the nucleus.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属(Eucalyptus)的10 000条EST序列进行分析, 在其中的1 499条序列上共发现1 775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外, 还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关; 微卫星的丰度与重复单元长度也呈负相关(三碱基重复微卫星除外)。在桉树EST序列中, 重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面, 桉树EST序列与杨树(Populus trichocarpa)基因组注释的转录序列随重复单元长度的变化呈现出相同的规律, 但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低, 推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计, 并对设计的引物进行PCR检测, 结果表明所设计的引物具有极高的扩增成功率。     

Post-transcriptional regulation of RNase-L expression is mediated by the 3'-untranslated region of its mRNA

RNase-L mediates critical cellular functions including antiviral, pro-apoptotic, and tumor suppressive activities; accordingly, its expression must be tightly regulated. Little is known about the control of RNASEL expression; therefore, we examined the potential regulatory role of a conserved 3'-untranslated region (3'-UTR) in its mRNA. The 3'-UTR mediated a potent decrease in the stability of RNase-L mRNA, and of a chimeric beta-globin-3'-UTR reporter mRNA. AU-rich elements (AREs) are cis-acting regulatory regions that modulate mRNA stability. Eight AREs were identified in the RNase-L 3'-UTR, and deletion analysis identified positive and negative regulatory regions associated with distinct AREs. In particular, AREs 7 and 8 served a strong positive regulatory function. HuR is an ARE-binding protein that stabilizes ARE-containing mRNAs, and a predicted HuR binding site was identified in the region comprising AREs 7 and 8. Co-transfection of HuR and RNase-L enhanced RNase-L expression and mRNA stability in a manner that was dependent on this 3'-UTR region. Immunoprecipitation demonstrated that RNase-L mRNA associates with a HuR containing complex in intact cells. Activation of endogenous HuR by cell stress, or during myoblast differentiation, increased RNase-L expression, suggesting that RNase-L mRNA is a physiologic target for HuR. HuR-dependent regulation of RNase-L enhanced its antiviral activity demonstrating the functional significance of this regulation. These findings identify a novel mechanism of RNase-L regulation mediated by its 3'-UTR.     

An unusually low microsatellite mutation rate in Dictyostelium discoideum, an organism with unusually abundant microsatellites

The genome of the social amoeba Dictyostelium discoideum is known to have a very high density of microsatellite repeats, including thousands of triplet microsatellite repeats in coding regions that apparently code for long runs of single amino acids. We used a mutation accumulation study to see if unusually high microsatellite mutation rates contribute to this pattern. There was a modest bias toward mutations that increase repeat number, but because upward mutations were smaller than downward ones, this did not lead to a net average increase in size. Longer microsatellites had higher mutation rates than shorter ones, but did not show greater directional bias. The most striking finding is that the overall mutation rate is the lowest reported for microsatellites: approximately 1 x 10(-6) for 10 dinucleotide loci and 6 x 10(-6) for 52 trinucleotide loci (which were longer). High microsatellite mutation rates therefore do not explain the high incidence of microsatellites. The causal relation may in fact be reversed, with low mutation rates evolving to protect against deleterious fitness effects of mutation at the numerous microsatellites.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属（Eucalyptus）的10000条EST序列进行分析,在其中的1499条序列上共发现1775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外,还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关;微卫星的丰度与重复单元长度也呈负相关（三碱基重复微卫星除外）。在桉树EST序列中,重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面,桉树EST序列与杨树（Populus trichocarpa）基因组注释的转录序列随重复单元长度的变化呈现出相同的规律,但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低,推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计,并对设计的引物进行PCR检测,结果表明所设计的引物具有极高的扩增成功率。     

Evolution of chloroplast mononucleotide microsatellites in Arabidopsis thaliana

The level of variation and the mutation rate were investigated in an empirical study of 244 chloroplast microsatellites in 15 accessions of Arabidopsis thaliana. In contrast to SNP variation, microsatellite variation in the chloroplast was found to be common, although less common than microsatellite variation in the nucleus. No microsatellite variation was found in coding regions of the chloroplast. To evaluate different models of microsatellite evolution as possible explanations for the observed pattern of variation, the length distribution of microsatellites in the published DNA sequence of the A. thaliana chloroplast was subsequently used. By combining information from these two analyses we found that the mode of evolution of the chloroplast mononucleotide microsatellites was best described by a linear relation between repeat length and mutation rate, when the repeat lengths exceeded about 7 bp. This model can readily predict the variation observed in non-coding chloroplast DNA. It was found that the number of uninterrupted repeat units had a large impact on the level of chloroplast microsatellite variation. No other factors investigated—such as the position of a locus within the chromosome, or imperfect repeats—appeared to affect the variability of chloroplast microsatellites. By fitting the slippage models to the Genbank sequence of chromosome 1, we show that the difference between microsatellite variation in the nucleus and the chloroplast is largely due to differences in slippage rate. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.