• Defects in THO/TREX-2 function cause accumulation of novel cytoplasmic mRNP granules that can be cleared by autophagy

      Eshleman, Nichole; Liu, Guangbo; McGrath, Kaitlyn; Parker, Roy; Buchan, J Ross; Univ Arizona, Dept Mol & Cellular Biol (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2016-06-01)
      The nuclear THO and TREX-2 complexes are implicated in several steps of nuclear mRNP biogenesis, including transcription, 3′ end processing and export. In a recent genomic microscopy screen in Saccharomyces cerevisiae for mutants with constitutive stress granules, we identified that absence of THO and TREX-2 complex subunits leads to the accumulation of Pab1-GFP in cytoplasmic foci. We now show that these THO/TREX-2 mutant induced foci (“TT foci”) are not stress granules but instead are a mRNP granule containing poly(A)+ mRNA, some mRNP components also found in stress granules, as well several proteins involved in mRNA 3′ end processing and export not normally seen in stress granules. In addition, TT foci are resistant to cycloheximide-induced disassembly, suggesting the presence of mRNPs impaired for entry into translation. THO mutants also exhibit defects in normal stress granule assembly. Finally, our data also suggest that TT foci are targeted by autophagy. These observations argue that defects in nuclear THO and TREX-2 complexes can affect cytoplasmic mRNP function by producing aberrant mRNPs that are exported to cytosol, where they accumulate in TT foci and ultimately can be cleared by autophagy. This identifies a novel mechanism of quality control for aberrant mRNPs assembled in the nucleus.
    • Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging

      Jeffries, Aaron R; Maroofian, Reza; Salter, Claire G; Chioza, Barry A; Cross, Harold E; Patton, Michael A; Dempster, Emma; Temple, I Karen; Mackay, Deborah J G; Rezwan, Faisal I; et al. (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2019-07)
      Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha (DNMT3A) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in DNMT3A c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo DNMT3A variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: NSD1 Sotos syndrome overgrowth disorder and KMT2D Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders.
    • Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2

      Keppetipola, Niroshika M; Yeom, Kyu-Hyeon; Hernandez, Adrian L; Bui, Tessa; Sharma, Shalini; Black, Douglas L; Univ Arizona, Dept Basic Med Sci, Coll Med (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2016-06-10)
      Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous polypyrimidine tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.
    • Rat BodyMap transcriptomes reveal unique circular RNA features across tissue types and developmental stages

      Zhou, Tong; Xie, Xueying; Li, Musheng; Shi, Junchao; Zhou, Jin J; Knox, Kenneth S; Wang, Ting; Chen, Qi; Gu, Wanjun; Univ Arizona, Dept Epidemiol & Biostat; et al. (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2018-08-09)
      Circular RNAs (circRNAs) are a novel class of regulatory RNAs. Here, we present a comprehensive investigation of circRNA expression profiles across 11 tissues and four developmental stages in rats, along with cross-species analyses in humans and mice. Although the expression of circRNAs is positively correlated with that of cognate mRNAs, highly expressed genes tend to splice a larger fraction of circular transcripts. Moreover, circRNAs exhibit higher tissue specificity than cognate mRNAs. Intriguingly, while we observed a monotonic increase of circRNA abundance with age in the rat brain, we further discovered a dynamic, age-dependent pattern of circRNA expression in the testes that is characterized by a dramatic increase with advancing stages of sexual maturity and a decrease with aging. The age-sensitive testicular circRNAs are highly associated with spermatogenesis, independent of cognate mRNA expression. The tissue/age implications of circRNAs suggest that they present unique physiological functions rather than simply occurring as occasional by-products of gene transcription.
    • Repeat associated mechanisms of genome evolution and function revealed by the Mus caroli and Mus pahari genomes

      Thybert, David; Roller, Maša; Navarro, Fábio C.P.; Fiddes, Ian; Streeter, Ian; Feig, Christine; Martin-Galvez, David; Kolmogorov, Mikhail; Janoušek, Václav; Akanni, Wasiu; et al. (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2018-03-21)
      Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 and 6 million yr ago, but that are absent in the Hominidae. Hominidae show between four- and sevenfold lower rates of nucleotide change and feature turnover in both neutral and functional sequences, suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. Recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli, which resulted in thousands of novel, species-specific CTCF binding sites. Our results show that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.
    • Structural variants in 3000 rice genomes

      Fuentes, Roven Rommel; Chebotarov, Dmytro; Duitama, Jorge; Smith, Sean; De la Hoz, Juan Fernando; Mohiyuddin, Marghoob; Wing, Rod A; McNally, Kenneth L; Tatarinova, Tatiana; Grigoriev, Andrey; et al. (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2019-04-16)
      Investigation of large structural variants (SVs) is a challenging yet important task in understanding trait differences in highly repetitive genomes. Combining different bioinformatic approaches for SV detection, we analyzed whole-genome sequencing data from 3000 rice genomes and identified 63 million individual SV calls that grouped into1.5 million allelic variants. We found enrichment of long SVs in promoters and an excess of shorter variants in 5' UTRs. Across the rice genomes, we identified regions of high SV frequency enriched in stress response genes. We demonstrated how SVs may help in finding causative variants in genome-wide association analysis. These new insights into rice genome biology are valuable for understanding the effects SVs have on gene function, with the prospect of identifying novel agronomically important alleles that can be utilized to improve cultivated rice.