The complete mitochondrial genome of Calappa bilineata: The first representative from the family Calappidae and its phylogenetic position within Brachyura.

In this study, we determined the complete mitogenome sequence of Calappa bilineata, which is the first mitogenome of Calappidae up to now. The total length is 15,606 bp and includes 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs and one control region. The genome composition is highly A + T biased (68.7%), and exhibits a negative AT-skew (-0.010) and GC-skew (-0.267). As with other invertebrate mitogenomes, the PCGs start with the standard ATN and stop with the standard TAN codons or incomplete T. Phylogenetic analysis showed that C. bilineata was most closely related to Matuta planipes (Matutidae), and these two species formed a sister clade, constituting a Calappoidea group and forming a sister clade with part of Eriphioidea. The existence of the polyphyletic families raised doubts over the traditional classification system. These results will help to better understand the features of the C. bilineata mitogenome and lay foundation for further evolutionary relationships within Brachyura.

Novel look at DNA and life-Symmetry as evolutionary forcing.

After explanation of the Chargaff´s first parity rule in terms of the Watson-Crick base-pairing between the two DNA strands, the Chargaff´s second parity rule for each strand of DNA (also named strand symmetry), which cannot be explained by Watson-Crick base-pairing only, is still a challenging issue already fifty years. We show that during evolution DNA preserves its identity in the form of quadruplet A+T and C+G rich matrices based on purine-pyrimidine mirror symmetries of trinucleotides. Identical symmetries are present in our classification of trinucleotides and the genetic code table. All eukaryotes and almost all prokaryotes (bacteria and archaea) have quadruplet mirror symmetries in structural form and frequencies following the principle of Chargaff's second parity rule and Natural symmetry law of DNA creation and conservation. Some rare symbionts have mirror symmetry only in their structural form within each DNA strand. Based on our matrix analysis of closely related species, humans and Neanderthals, we find that the circular cycle of inverse proportionality between trinucleotides preserves identical relative frequencies of trinucleotides in each quadruplet and in the whole genome. According to our calculations, a change in frequencies in quadruplet matrices could lead to the creation of new species. Violation of quadruplet symmetries is practically inconsistent with life. DNA symmetries provide a key for understanding the restriction of disorder (entropy) due to mutations in the evolution of DNA.

BLM prevents instability of structure-forming DNA sequences at common fragile sites.

Genome instability often arises at common fragile sites (CFSs) leading to cancer-associated chromosomal rearrangements. However, the underlying mechanisms of how CFS protection is achieved is not well understood. We demonstrate that BLM plays an important role in the maintenance of genome stability of structure-forming AT-rich sequences derived from CFSs (CFS-AT). BLM deficiency leads to increased DSB formation and hyper mitotic recombination at CFS-AT and induces instability of the plasmids containing CFS-AT. We further showed that BLM is required for suppression of CFS breakage upon oncogene expression. Both helicase activity and ATR-mediated phosphorylation of BLM are important for preventing genetic instability at CFS-AT sequences. Furthermore, the role of BLM in protecting CFS-AT is not epistatic to that of FANCM, a translocase that is involved in preserving CFS stability. Loss of BLM helicase activity leads to drastic decrease of cell viability in FANCM deficient cells. We propose that BLM and FANCM utilize different mechanisms to remove DNA secondary structures forming at CFS-AT on replication forks, thereby preventing DSB formation and maintaining CFS stability.

Binding of high mobility group A proteins to the mammalian genome occurs as a function of AT-content.

Genomic location can inform on potential function and recruitment signals for chromatin-associated proteins. High mobility group (Hmg) proteins are of similar size as histones with Hmga1 and Hmga2 being particularly abundant in replicating normal tissues and in cancerous cells. While several roles for Hmga proteins have been proposed we lack a comprehensive description of their genomic location as a function of chromatin, DNA sequence and functional domains. Here we report such a characterization in mouse embryonic stem cells in which we introduce biotin-tagged constructs of wild-type and DNA-binding domain mutants. Comparative analysis of the genome-wide distribution of Hmga proteins reveals pervasive binding, a feature that critically depends on a functional DNA-binding domain and which is shared by both Hmga proteins. Assessment of the underlying queues instructive for this binding modality identifies AT richness, defined as high frequency of A or T bases, as the major criterion for local binding. Additionally, we show that other chromatin states such as those linked to cis-regulatory regions have little impact on Hmga binding both in stem and differentiated cells. As a consequence, Hmga proteins are preferentially found at AT-rich regions such as constitutively heterochromatic regions but are absent from enhancers and promoters arguing for a limited role in regulating individual genes. In line with this model, we show that genetic deletion of Hmga proteins in stem cells causes limited transcriptional effects and that binding is conserved in neuronal progenitors. Overall our comparative study describing the in vivo binding modality of Hmga1 and Hmga2 identifies the proteins' preference for AT-rich DNA genome-wide and argues against a suggested function of Hmga at regulatory regions. Instead we discover pervasive binding with enrichment at regions of higher AT content irrespective of local variation in chromatin modifications.

DNA helix: the importance of being AT-rich.

The AT-rich DNA is mostly associated with condensed chromatin, whereas the GC-rich sequence is preferably located in the dispersed chromatin. The AT-rich genes are prone to be tissue-specific (silenced in most tissues), while the GC-rich genes tend to be housekeeping (expressed in many tissues). This paper reports another important property of DNA base composition, which can affect repertoire of genes with high AT content. The GC-rich sequence is more liable to mutation. We found that Spearman correlation between human gene GC content and mutation probability is above 0.9. The change of base composition even in synonymous sites affects mutation probability of nonsynonymous sites and thus of encoded proteins. There is a unique type of housekeeping genes, which are especially unsafe when prone to mutation. Natural selection which usually removes deleterious mutations, in the case of these genes only increases the hazard because it can descend to suborganismal (cellular) level. These are cell cycle-related genes. In accordance with the proposed concept, they have low GC content of synonymous sites (despite them being housekeeping). The gene-centred protein interaction enrichment analysis (PIEA) showed the core clusters of genes whose interactants are modularly enriched in genes with AT-rich synonymous codons. This interconnected network is involved in double-strand break repair, DNA integrity checkpoints and chromosome pairing at mitosis. The damage of these genes results in genome and chromosome instability leading to cancer and other 'error catastrophes'. Reducing the nonsynonymous mutations, the usage of AT-rich synonymous codons can decrease probability of cancer by above 20-fold.

DNA flap creation by the RarA/MgsA protein of Escherichia coli.

We identify a novel activity of the RarA (also MgsA) protein of Escherichia coli, demonstrating that this protein functions at DNA ends to generate flaps. A AAA+ ATPase in the clamp loader clade, RarA protein is part of a highly conserved family of DNA metabolism proteins. We demonstrate that RarA binds to double-stranded DNA in its ATP-bound state and single-stranded DNA in its apo state. RarA ATPase activity is stimulated by single-stranded DNA gaps and double-stranded DNA ends. At these double-stranded DNA ends, RarA couples the energy of ATP binding and hydrolysis to separating the strands of duplex DNA, creating flaps. We hypothesize that the creation of a flap at the site of a leading strand discontinuity could, in principle, allow DnaB and the associated replisome to continue DNA synthesis without impediment, with leading strand re-priming by DnaG. Replication forks could thus be rescued in a manner that does not involve replisome disassembly or reassembly, albeit with loss of one of the two chromosomal products of a replication cycle.

The Plastid Genome of Polytoma uvella Is the Largest Known among Colorless Algae and Plants and Reflects Contrasting Evolutionary Paths to Nonphotosynthetic Lifestyles.

The loss of photosynthesis is frequently associated with parasitic or pathogenic lifestyles, but it also can occur in free-living, plastid-bearing lineages. A common consequence of becoming nonphotosynthetic is the reduction in size and gene content of the plastid genome. In exceptional circumstances, it can even result in the complete loss of the plastid DNA (ptDNA) and its associated gene expression system, as reported recently in several lineages, including the nonphotosynthetic green algal genus Polytomella Closely related to Polytomella is the polyphyletic genus Polytoma, the members of which lost photosynthesis independently of Polytomella Species from both genera are free-living organisms that contain nonphotosynthetic plastids, but unlike Polytomella, Polytoma members have retained a genome in their colorless plastid. Here, we present the plastid genome of Polytoma uvella: to our knowledge, the first report of ptDNA from a nonphotosynthetic chlamydomonadalean alga. The P. uvella ptDNA contains 25 protein-coding genes, most of which are related to gene expression and none are connected to photosynthesis. However, despite its reduced coding capacity, the P. uvella ptDNA is inflated with short repeats and is tens of kilobases larger than the ptDNAs of its closest known photosynthetic relatives, Chlamydomonas leiostraca and Chlamydomonas applanata In fact, at approximately 230 kb, the ptDNA of P. uvella represents the largest plastid genome currently reported from a nonphotosynthetic alga or plant. Overall, the P. uvella and Polytomella plastid genomes reveal two very different evolutionary paths following the loss of photosynthesis: expansion and complete deletion, respectively. We hypothesize that recombination-based DNA-repair mechanisms are at least partially responsible for the different evolutionary outcomes observed in such closely related nonphotosynthetic algae.

The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II)2Cys6 transcription factor BcBot6.

Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G+C/A+T-equilibrated regions that contain the Bcbot genes alternate with A+T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)2Cys6 putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the ΔBcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BOT synthesis and of its ecological role in the B. cinerea life cycle.

Mitochondrial genomes of praying mantises (Dictyoptera, Mantodea): rearrangement, duplication, and reassignment of tRNA genes.

Insect mitochondrial genomes (mitogenomes) contain a conserved set of 37 genes for an extensive diversity of lineages. Previously reported dictyopteran mitogenomes share this conserved mitochondrial gene arrangement, although surprisingly little is known about the mitogenome of Mantodea. We sequenced eight mantodean mitogenomes including the first representatives of two families: Hymenopodidae and Liturgusidae. Only two of these genomes retain the typical insect gene arrangement. In three Liturgusidae species, the trnM genes have translocated. Four species of mantis (Creobroter gemmata, Mantis religiosa, Statilia sp., and Theopompa sp.-HN) have multiple identical tandem duplication of trnR, and Statilia sp. additionally includes five extra duplicate trnW. These extra trnR and trnW in Statilia sp. are erratically arranged and form another novel gene order. Interestingly, the extra trnW is converted from trnR by the process of point mutation at anticodon, which is the first case of tRNA reassignment for an insect. Furthermore, no significant differences were observed amongst mantodean mitogenomes with variable copies of tRNA according to comparative analysis of codon usage. Combined with phylogenetic analysis, the characteristics of tRNA only possess limited phylogenetic information in this research. Nevertheless, these features of gene rearrangement, duplication, and reassignment provide valuable information toward understanding mitogenome evolution in insects.

Characterization of the complete mitochondrial genome of the storage mite pest Tyrophagus longior (Gervais) (Acari: Acaridae) and comparative mitogenomic analysis of four acarid mites.

Mites of the genus Tyrophagus are economically important polyphagous pest commonly living on stored products and also responsible for allergic reactions to humans. Complete mitochondrial genomes (mitogenomes) and the gene features therein are widely used as molecular markers in the study of population genetics, phylogenetics as well as molecular evolution. However, scarcity on the sequence data has greatly impeded the studies in these areas pertaining to the Acari (mites and ticks). Information on the Tyrophagus mitogenomes is quite critical for phylogenetic evaluation and molecular evolution of the mitogenomes within Acariformes. Herein, we reported the complete mitogenome of the allergenic acarid storage mite Tyrophagus longior (Astigmata: Acaridae), an important member of stored food pests, and compared with those of other three acarid mites. The complete mitogenome of T. longior was a circular molecule of 13,271 bp. Unexpectedly, only 19 transfer RNA genes (tRNAs) were present, lacking trnF, trnS1 and trnQ. Furthermore, it also contained 13 protein-coding genes (PCGs) and 2 genes for rRNA (rrnS and rrnL) commonly detected in metazoans. The four mitogenomes displayed similar characteristics with respect to the gene content, nucleotide comparison, and codon usages. Yet, the gene order of T. longior was different from that in other Acari. The J-strands of the four mitogenomes possessed high A+T content (67.4-70.0%), and exhibited positive GC-skews and negative AT-skews. Most inferred tRNAs of T. longior were extremely truncated, lacking either a D- or T-arm, as found in other acarid mites. In T. longior mitogenome the A+T-rich region was just 50 bp in length and can be folded as a stable stem-loop structure, whereas in the region some structures of microsatellite-like (AT)n and palindromic sequences was not present. Besides, reconstructing of the phylogenetic relationship based on concatenated amino acid sequences of 13 PCGs supported that monophyly of the family Acaridae and the order Astigmata, to which the former belongs. Our results were consistent with the traditional classifications.

The mitochondrial genome of the multicolored Asian lady beetle Harmonia axyridis (Pallas) and a phylogenetic analysis of the Polyphaga (Insecta: Coleoptera).

Here, we report the mitochondrial genome sequence of the multicolored Asian lady beetle Harmonia axyridis (Pallas, 1773) (Coleoptera: Coccinellidae) (GenBank accession No. KR108208). This is the first species with sequenced mitochondrial genome from the genus Harmonia. The current length with partitial A + T-rich region of this mitochondrial genome is 16,387 bp. All the typical genes were sequenced except the trnI and trnQ. As in most other sequenced mitochondrial genomes of Coleoptera, there is no re-arrangement in the sequenced region compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN codons. Five, five and three protein-coding genes stop with termination codon TAA, TA and T, respectively. Phylogenetic analysis using Bayesian method based on the first and second codon positions of the protein-coding genes supported that the Scirtidae is a basal lineage of Polyphaga. The Harmonia and the Coccinella form a sister lineage. The monophyly of Staphyliniformia, Scarabaeiformia and Cucujiformia was supported. The Buprestidae was found to be a sister group to the Bostrichiformia.

Sequence-specific recognition of DNA minor groove by an NIR-fluorescence switch-on probe and its potential applications.

In molecular biology, understanding the functional and structural aspects of DNA requires sequence-specific DNA binding probes. Especially, sequence-specific fluorescence probes offer the advantage of real-time monitoring of the conformational and structural reorganization of DNA in living cells. Herein, we designed a new class of D2A (one-donor-two-acceptor) near-infrared (NIR) fluorescence switch-on probe named quinone cyanine-dithiazole ( QCY-DT: ) based on the distinctive internal charge transfer (ICT) process for minor groove recognition of AT-rich DNA. Interestingly, QCY-DT: exhibited strong NIR-fluorescence enhancement in the presence of AT-rich DNA compared to GC-rich and single-stranded DNAs. We show sequence-specific minor groove recognition of QCY-DT: for DNA containing 5'-AATT-3' sequence over other variable (A/T)4 sequences and local nucleobase variation study around the 5'-X(AATT)Y-3' recognition sequence revealed that X = A and Y = T are the most preferable nucleobases. The live cell imaging studies confirmed mammalian cell permeability, low-toxicity and selective staining capacity of nuclear DNA without requiring RNase treatment. Further, Plasmodium falciparum with an AT-rich genome showed specific uptake with a reasonably low IC50 value (<4 µM). The ease of synthesis, large Stokes shift, sequence-specific DNA minor groove recognition with switch-on NIR-fluorescence, photostability and parasite staining with low IC50 make QCY-DT: a potential and commercially viable DNA probe.

Free energy landscape and transition pathways from Watson-Crick to Hoogsteen base pairing in free duplex DNA.

Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson-Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine-thymine (A-T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events.

Insulin acutely triggers transcription of Slc2a4 gene: participation of the AT-rich, E-box and NFKB-binding sites.

AIMS: The insulin-sensitive glucose transporter protein GLUT4 (solute carrier family 2 member 4 (Slc2a4) gene) plays a key role in glycemic homeostasis. Decreased GLUT4 expression is a current feature in insulin resistant conditions such as diabetes, and the restoration of GLUT4 content improves glycemic control. This study investigated the effect of insulin upon Slc2a4/GLUT4 expression, focusing on the AT-rich element, E-box and nuclear factor NF-kappa-B (NFKB) site. MAIN METHODS: Rat soleus muscles were incubated during 180 min with insulin, added or not with wortmannin (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PI3K)-inhibitor), ML9 (serine/threonine protein kinase (AKT) inhibitor) and tumor necrosis factor (TNF, GLUT4 repressor), and processed for analysis of GLUT4 protein (Western blotting); Slc2a4, myocyte enhancer factor 2a/d (Mef2a/d), hypoxia inducible factor 1a (Hif1a), myogenic differentiation 1 (Myod1) and nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfkb1) messenger ribonucleic acids (mRNAs) (polymerase chain reaction (PCR)); and AT-rich- (myocyte-specific enhancer factor 2 (MEF2)-binding site), E-box- (hypoxia inducible factor 1 alpha (HIF1A)- and myoblast determination protein 1 (MYOD1)-binding site), and NFKB-binding activity (electrophoretic mobility assay). KEY FINDINGS: Insulin increased Slc2a4 mRNA expression (140%) and nuclear proteins binding to AT-rich and E-box elements (~90%), all effects were prevented by wortmannin and ML9. Insulin also increased Mef2a/d and Myod1 mRNA expression, suggesting the participation of these transcriptional factors in the Slc2a4 enhancing effect. Conversely, insulin decreased Nfkb1 mRNA expression and protein binding to the NFKB-site (~50%). Furthermore, TNF-induced inhibition of GLUT4 expression (~40%) was prevented by insulin in an NFKB-binding repressing mechanism. GLUT4 protein paralleled the Slc2a4 mRNA regulations. SIGNIFICANCE: Insulin enhances the Slc2a4/GLUT4 expression in the skeletal muscle by activating AT-rich and E-box elements, in a PI3K/AKT-dependent mechanism, and repressing NFKB-site activity as well. These results unravel how post-prandial increase of insulin may guarantee GLUT4 expression, and how the insulin signaling impairment can participate in insulin resistance-induced repression of GLUT4.

Crystal structure of the ubiquitin-like domain-CUT repeat-like tandem of special AT-rich sequence binding protein 1 (SATB1) reveals a coordinating DNA-binding mechanism.

SATB1 is essential for T-cell development and growth and metastasis of multitype tumors and acts as a global chromatin organizer and gene expression regulator. The DNA binding ability of SATB1 plays vital roles in its various biological functions. We report the crystal structure of the N-terminal module of SATB1. Interestingly, this module contains a ubiquitin-like domain (ULD) and a CUT repeat-like (CUTL) domain (ULD-CUTL tandem). Detailed biochemical experiments indicate that the N terminus of SATB1 (residues 1-248, SATB1((1-248))), including the extreme 70 N-terminal amino acids, and the ULD-CUTL tandem bind specifically to DNA targets. Our results show that the DNA binding ability of full-length SATB1 requires the contribution of the CUTL domain, as well as the CUT1-CUT2 tandem domain and the homeodomain. These findings may reveal a multiple-domain-coordinated mechanism whereby SATB1 recognizes DNA targets.

Analysis of the t(3;8) of hereditary renal cell carcinoma: a palindrome-mediated translocation.

It has emerged that palindrome-mediated genomic instability generates DNA-based rearrangements. The presence of palindromic AT-rich repeats (PATRRs) at the translocation breakpoints suggested a palindrome-mediated mechanism in the generation of several recurrent constitutional rearrangements: the t(11;22), t(17;22), and t(8;22). To date, all reported PATRR-mediated translocations include the PATRR on chromosome 22 (PATRR22) as a translocation partner. Here, the constitutional rearrangement, t(3;8)(p14.2;q24.1), segregating with renal cell carcinoma in two families, is examined. The chromosome 8 breakpoint lies in PATRR8 in the first intron of the RNF139 (TRC8) gene, whereas the chromosome 3 breakpoint is located in an AT-rich palindromic sequence in intron 3 of the FHIT gene (PATRR3). Thus, the t(3;8) is the first PATRR-mediated, recurrent, constitutional translocation that does not involve PATRR22. Furthermore, we detect de novo translocations similar to the t(11;22) and t(8;22), involving PATRR3 in normal sperm. The breakpoint on chromosome 3 is in proximity to FRA3B, the most common fragile site in the human genome and a site of frequent deletions in tumor cells. However, the lack of involvement of PATRR3 sequence in numerous FRA3B-related deletions suggests that there are several different DNA sequence-based etiologies responsible for chromosome 3p14.2 genomic rearrangements.

Somatic hypermutation at A/T-rich oligonucleotide substrates shows different strand polarities in Ung-deficient or -proficient backgrounds.

A/T mutations at immunoglobulin loci are introduced by DNA polymerase η (Polη) during an Msh2/6-dependent repair process which results in A's being mutated 2-fold more often than T's. This patch synthesis is initiated by a DNA incision event whose origin is still obscure. We report here the analysis of A/T oligonucleotide mutation substrates inserted at the heavy chain locus, including or not including internal C's or G's. Surprisingly, the template composed of only A's and T's was highly mutated over its entire 90-bp length, with a 2-fold decrease in mutation from the 5' to the 3' end and a constant A/T ratio of 4. These results imply that Polη synthesis was initiated from a break in the 5'-flanking region of the substrate and proceeded over its entire length. The A/T bias was strikingly altered in an Ung(-/-) background, which provides the first experimental evidence supporting a concerted action of Ung and Msh2/6 pathways to generate mutations at A/T bases. New analysis of Pms2(-/-) animals provided a complementary picture, revealing an A/T mutation ratio of 4. We therefore propose that Ung and Pms2 may exert a mutual backup function for the DNA incision that promotes synthesis by Polη, each with a distinct strand bias.

Somatic mutations found in the healthy blood compartment of a 115-yr-old woman demonstrate oligoclonal hematopoiesis.

The somatic mutation burden in healthy white blood cells (WBCs) is not well known. Based on deep whole-genome sequencing, we estimate that approximately 450 somatic mutations accumulated in the nonrepetitive genome within the healthy blood compartment of a 115-yr-old woman. The detected mutations appear to have been harmless passenger mutations: They were enriched in noncoding, AT-rich regions that are not evolutionarily conserved, and they were depleted for genomic elements where mutations might have favorable or adverse effects on cellular fitness, such as regions with actively transcribed genes. The distribution of variant allele frequencies of these mutations suggests that the majority of the peripheral white blood cells were offspring of two related hematopoietic stem cell (HSC) clones. Moreover, telomere lengths of the WBCs were significantly shorter than telomere lengths from other tissues. Together, this suggests that the finite lifespan of HSCs, rather than somatic mutation effects, may lead to hematopoietic clonal evolution at extreme ages.

Palindrome-mediated and replication-dependent pathogenic structural rearrangements within the NF1 gene.

Palindromic sequences can form hairpin structures or cruciform extrusions, which render them susceptible to genomic rearrangements. A 197-bp long palindromic AT-rich repeat (PATRR17) is located within intron 40 of the neurofibromatosis type 1 (NF1) gene (17q11.2). Through comprehensive NF1 analysis, we identified six unrelated patients with a rearrangement involving intron 40 (five deletions and one reciprocal translocation t(14;17)(q32;q11.2)). We hypothesized that PATRR17 may be involved in these rearrangements thereby causing NF1. Breakpoint cloning revealed that PATRR17 was indeed involved in all of the rearrangements. As microhomology was present at all breakpoint junctions of the deletions identified, and PATRR17 partner breakpoints were located within 7.1 kb upstream of PATRR17, fork stalling and template switching/microhomology-mediated break-induced replication was the most likely rearrangement mechanism. For the reciprocal translocation case, a 51 bp insertion at the translocation breakpoints mapped to a short sequence within PATRR17, proximal to the breakpoint, suggesting a multiple stalling and rereplication process, in contrast to previous studies indicating a purely replication-independent mechanism for PATRR-mediated translocations. In conclusion, we show evidence that PATRR17 is a hotspot for pathogenic intragenic deletions within the NF1 gene and suggest a novel replication-dependent mechanism for PATRR-mediated translocation.

Knockdown of Hnrnpa0, a del(5q) gene, alters myeloid cell fate in murine cells through regulation of AU-rich transcripts.

The control of mRNA stability plays a central role in orchestrating gene-regulatory networks in hematopoietic cell growth, differentiation and tumorigenesis. HNRNPA0, which encodes an RNA-binding protein shown to regulate transcript stability via binding to the AU-rich elements of mRNAs, is located within the commonly deleted segment of 5q31.2 in myeloid neoplasms with a del(5q), and is expressed at haploinsufficient levels in these patients. We show that HNRNPA0 is normally highly expressed in hematopoietic stem cells and exhibits dynamic changes in expression during the course of differentiation. To model HNRNPA0 haploinsufficiency, we used RNAi interference in primary murine cells and an experimental cell system, and found that reduced Hnrnpa0 expression leads to a shift from monocytic towards granulocytic differentiation. Microarray-based global expression profiling revealed that Hnrnpa0 knockdown disproportionally impacts AU-rich containing transcripts and alters expression of myeloid specification genes. In therapy-related myeloid neoplasms with a del(5q), AU-rich containing mRNAs are enriched in transcripts that encode proteins associated with increased growth and proliferation. Our findings implicate haploinsufficiency of HNRNPA0 as one of the key initiating mutations in the pathogenesis of myeloid neoplasms with a del(5q), and suggest that therapies that target AU-rich elements warrant consideration in efforts to develop new mechanism-based treatment strategies.