Reliance of Host-Encoded Regulators of Retromobility on Ty1 Promoter Activity or Architecture.

The Ty1 retrotransposon family is maintained in a functional but dormant state by its host, Saccharomyces cerevisiae. Several hundred RHF and RTT genes encoding co-factors and restrictors of Ty1 retromobility, respectively, have been identified. Well-characterized examples include MED3 and MED15, encoding subunits of the Mediator transcriptional co-activator complex; control of retromobility by Med3 and Med15 requires the Ty1 promoter in the U3 region of the long terminal repeat. To characterize the U3-dependence of other Ty1 regulators, we screened a library of 188 known rhf and rtt mutants for altered retromobility of Ty1his3AI expressed from the strong, TATA-less TEF1 promoter or the weak, TATA-containing U3 promoter. Two classes of genes, each including both RHFs and RTTs, were identified. The first class comprising 82 genes that regulated Ty1his3AI retromobility independently of U3 is enriched for RHF genes that restrict the G1 phase of the cell cycle and those involved in transcriptional elongation and mRNA catabolism. The second class of 51 genes regulated retromobility of Ty1his3AI driven only from the U3 promoter. Nineteen U3-dependent regulators (U3DRs) also controlled retromobility of Ty1his3AI driven by the weak, TATA-less PSP2 promoter, suggesting reliance on the low activity of U3. Thirty-one U3DRs failed to modulate P PSP2 -Ty1his3AI retromobility, suggesting dependence on the architecture of U3. To further investigate the U3-dependency of Ty1 regulators, we developed a novel fluorescence-based assay to monitor expression of p22-Gag, a restriction factor expressed from the internal Ty1i promoter. Many U3DRs had minimal effects on levels of Ty1 RNA, Ty1i RNA or p22-Gag. These findings uncover a role for the Ty1 promoter in integrating signals from diverse host factors to modulate Ty1 RNA biogenesis or fate.

Border collies of the genome: domestication of an autonomous retrovirus-like transposon.

Retrotransposons often spread rapidly through eukaryotic genomes until they are neutralized by host-mediated silencing mechanisms, reduced by recombination and mutation, and lost or transformed into benevolent entities. But the Ty1 retrotransposon appears to have been domesticated to guard the genome of Saccharomyces cerevisiae.

Meeting report: mobile genetic elements and genome plasticity 2018.

The Mobile Genetic Elements and Genome Plasticity conference was hosted by Keystone Symposia in Santa Fe, NM USA, February 11-15, 2018. The organizers were Marlene Belfort, Evan Eichler, Henry Levin and Lynn Maquat. The goal of this conference was to bring together scientists from around the world to discuss the function of transposable elements and their impact on host species. Central themes of the meeting included recent innovations in genome analysis and the role of mobile DNA in disease and evolution. The conference included 200 scientists who participated in poster presentations, short talks selected from abstracts, and invited talks. A total of 58 talks were organized into eight sessions and two workshops. The topics varied from mechanisms of mobilization, to the structure of genomes and their defense strategies to protect against transposable elements.

Characterization of a novel HLA-C allele, HLA-C*04:288, in an Italian patient.

The novel HLA-C*04:288 differs from HLA-C*04:01:01:06 by a single nucleotide substitution in exon 2.

The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters.

The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting non-essential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility do not appear to effect genomic Ty1 RNA levels, Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.

Identification of the new HLA-DPB1 allele, DPB1*647:01, in an Italian patient with leukemia.

A novel HLA-DPB1 allele, named HLA-DPB1*647:01, identified in a leukemia patient.

Identification of two new HLA class I alleles in Italian bone marrow donors: A*31:125 and B*44:269.

Two novel alleles, HLA-A*31:125 and HLA-B*44:269, are described in Italian bone marrow donors.

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5' terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.

Paralog-Specific Functions of RPL7A and RPL7B Mediated by Ribosomal Protein or snoRNA Dosage in Saccharomyces cerevisiae.

Most ribosomal proteins in Saccharomyces cerevisiae are encoded by two paralogs that additively produce the optimal protein level for cell growth. Nonetheless, deleting one paralog of most ribosomal protein gene pairs results in a variety of phenotypes not observed when the other paralog is deleted. To determine whether paralog-specific phenotypes associated with deleting RPL7A or RPL7B stem from distinct functions or different levels of the encoded isoforms, the coding region and introns of one paralog, including an intron-embedded snoRNA (small nucleolar RNA) gene, were exchanged with that of the other paralog. Among mutants harboring a single native or chimeric RPL7 allele, expression from the RPL7A locus exceeded that from the RPL7B locus, and more Rpl7a was expressed from either locus than Rpl7b Phenotypic differences in tunicamycin sensitivity, ASH1 mRNA localization, and mobility of the Ty1 retrotransposon were strongly correlated with Rpl7 and ribosome levels, but not with the Rpl7 or snoRNA isoform expressed. Although Ty1 RNA is cotranslationally localized, depletion of Rpl7 minimally affected synthesis of Ty1 Gag protein, but strongly influenced Ty1 RNA localization. Unlike the other processes studied, Ty1 cDNA accumulation was influenced by both the level and isoform of Rpl7 or snoRNA expressed. These cellular processes had different minimal threshold values for Rpl7 and ribosome levels, but all were functional when isoforms of either paralog were expressed from the RPL7A locus or both RPL7 loci. This study illustrates the broad range of phenotypes that can result from depleting ribosomes to different levels.

Characterization of a novel HLA-B*18 variant allele, HLA-B*18:122, by genome sequencing.

The HLA-B*18:122 allele showed four nucleotide differences from HLA-B*18:01:01:01.

Characterization of a novel HLA-DRB1 allele, HLA-DRB1*13:215, in an Italian bone marrow donor.

One nucleotide substitution at residue 142 of the HLA-DRB1*13:01:01 results in a new allele, HLA-DRB1*13:215.

Glass-ceramic coated Mg-Ca alloys for biomedical implant applications.

Biodegradable metals and alloys are promising candidates for biomedical bone implant applications. However, due to the high rate of their biodegradation in human body environment, they should be coated with less reactive materials, such, for example, as bioactive glasses or glass-ceramics. Fort this scope, RKKP composition glass-ceramic coatings have been deposited on Mg-Ca(1.4wt%) alloy substrates by Pulsed Laser Deposition method, and their properties have been characterized by a number of techniques. The prepared coatings consist of hydroxyapatite and wollastonite phases, having composition close to that of the bulk target material used for depositions. The 100µm thick films are characterized by dense, compact and rough morphology. They are composed of a glassy matrix with various size (from micro- to nano-) granular inclusions. The average surface roughness is about 295±30nm due to the contribution of micrometric aggregates, while the roughness of the fine-texture particulates is approximately 47±4nm. The results of the electrochemical corrosion evaluation tests evidence that the RKKP coating improves the corrosion resistance of the Mg-Ca (1.4wt%) alloy in Simulated Body Fluid.

Identification of a new HLA-A*24 allele, A*24:309, in an Italian bone marrow donor.

A novel class I human leukocyte antigen allele HLA-A*24:309 is described.

Global Epitranscriptomics Profiling of RNA Post-Transcriptional Modifications as an Effective Tool for Investigating the Epitranscriptomics of Stress Response.

The simultaneous detection of all the post-transcriptional modifications (PTMs) that decorate cellular RNA can provide comprehensive information on the effects of changing environmental conditions on the entire epitranscriptome. To capture this type of information, we performed the analysis of ribonucleotide mixtures produced by hydrolysis of total RNA extracts from S. cerevisiae that was grown under hyperosmotic and heat shock conditions. Their global PTM profiles clearly indicated that the cellular responses to these types of stresses involved profound changes in the production of specific PTMs. The observed changes involved not only up-/down-regulation of typical PTMs, but also the outright induction of new ones that were absent under normal conditions, or the elimination of others that were normally present. Pointing toward the broad involvement of different classes of RNAs, many of the newly observed PTMs differed from those engaged in the known tRNA-based mechanism of translational recoding, which is induced by oxidative stress. Some of the expression effects were stress-specific, whereas others were not, thus suggesting that RNA PTMs may perform multifaceted activities in stress response, which are subjected to distinctive regulatory pathways. To explore their signaling networks, we implemented a strategy based on the systematic deletion of genes that connect established response genes with PTM biogenetic enzymes in a putative interactomic map. The results clearly identified PTMs that were under direct HOG control, a well-known protein kinase pathway involved in stress response in eukaryotes. Activation of this signaling pathway has been shown to result in the stabilization of numerous mRNAs and the induction of selected lncRNAs involved in chromatin remodeling. The fact that PTMs are capable of altering the activity of the parent RNAs suggest their possible participation in feedback mechanisms aimed at modulating the regulatory functions of such RNAs. This tantalizing hypothesis will be the object of future studies.

Characterization of a novel HLA-B allele (HLA-B*18:108) by intron-exon sequencing of the HLA-B locus.

HLA-B*18:108 has two nucleotide changes from B*18:01:01:02 at nt 430 where A → G and nt 431 where G → A.

The Ty1 LTR-Retrotransposon of Budding Yeast, Saccharomyces cerevisiae.

Long-terminal repeat (LTR)-retrotransposons generate a copy of their DNA (cDNA) by reverse transcription of their RNA genome in cytoplasmic nucleocapsids. They are widespread in the eukaryotic kingdom and are the evolutionary progenitors of retroviruses. The Ty1 element of the budding yeast Saccharomyces cerevisiae was the first LTR-retrotransposon demonstrated to mobilize through an RNA intermediate, and not surprisingly, is the best studied. The depth of our knowledge of Ty1 biology stems not only from the predominance of active Ty1 elements in the S. cerevisiae genome but also the ease and breadth of genomic, biochemical, and cell biology approaches available to study cellular processes in yeast. This review describes the basic structure of Ty1 and its gene products, the replication cycle, the rapidly expanding compendium of host cofactors known to influence retrotransposition, and the nature of Ty1's elaborate symbiosis with its host. Our goal is to illuminate the value of Ty1 as a paradigm to explore the biology of LTR-retrotransposons in multicellular organisms, where the low frequency of retrotransposition events presents a formidable barrier to investigations of retrotransposon biology.

The Ty1 LTR-retrotransposon of budding yeast, Saccharomyces cerevisiae.

Long-terminal repeat (LTR)-retrotransposons generate a copy of their DNA (cDNA) by reverse transcription of their RNA genome in cytoplasmic nucleocapsids. They are widespread in the eukaryotic kingdom and are the evolutionary progenitors of retroviruses [1]. The Ty1 element of the budding yeast Saccharomyces cerevisiae was the first LTR-retrotransposon demonstrated to mobilize through an RNA intermediate, and not surprisingly, is the best studied. The depth of our knowledge of Ty1 biology stems not only from the predominance of active Ty1 elements in the S. cerevisiae genome but also the ease and breadth of genomic, biochemical and cell biology approaches available to study cellular processes in yeast. This review describes the basic structure of Ty1 and its gene products, the replication cycle, the rapidly expanding compendium of host co-factors known to influence retrotransposition and the nature of Ty1's elaborate symbiosis with its host. Our goal is to illuminate the value of Ty1 as a paradigm to explore the biology of LTR-retrotransposons in multicellular organisms, where the low frequency of retrotransposition events presents a formidable barrier to investigations of retrotransposon biology.

Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons.

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected, and the functional consequences, have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51), but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD), a model of global brain ischemia, as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity, the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore, GDNF protected hippocampal neurons when present before, during or after OGD, and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF.

The influence of ictal cutaneous allodynia on the response to occipital transcutaneous electrical stimulation in chronic migraine and chronic tension-type headache: a randomized, sham-controlled study.

OBJECTIVE: The objective of this article is to determine whether cutaneous allodynia (CA) influences the response to treatment with occipital transcutaneous electrical stimulation (OTES) in chronic migraine (CM) and chronic tension-type headache (CTTH). METHODS: One hundred and sixty consecutive patients with CM or CTTH were randomized to be treated with real or sham OTES stimulation three times a day for two consecutive weeks. All patients completed the validated 12-item allodynia symptom checklist for assessing the presence and the severity of CA during headache attack. Primary end-point was change (≥50%) in number of monthly headache-free days. RESULTS: There was a significant difference in the percentage of responders in the real OTES compared with sham OTES group (p <0.001). Importantly, there was not a significant change of monthly headache-free days in the allodynic patients with CM and CTTH treated both with real and sham OTES, while the number of headache-free days per month was significantly reduced in the real (86%) but not in the sham group (7%) of non-allodynic patients with CTTH and CM. CONCLUSIONS: Severe CA is associated with decreased response to treatment with OTES in patients with CM and CTTH.

Identification and characterization of a novel HLA-A allele, HLA-A*68:105, by genomic sequencing.

A novel HLA-A allele, HLA-A*68:105, was detected by sequence-based typing (SBT) in an Italian bone marrow donor. It differs from HLA-A*68:01:02 at five nucleotides, three intronic, nt 699 T->G (intron 2), nt 705 T->C (intron 2) and nt 2770 G->A (intron 7), and two located in exon 3, at positions 726 A-G (codon 94 Ile->Val) and 733 T-G (codon 97 Arg->Met), respectively.