scholarly journals Transposon expression in the Drosophila brain is driven by neighboring genes and diversifies the neural transcriptome

2019 ◽  
Author(s):  
Christoph D. Treiber ◽  
Scott Waddell
Keyword(s):  
Neural Tissue ◽  
Cellular Gene ◽  
Nascent Transcript ◽  
Mrna Sequencing ◽  
Functional Differences ◽  
Cellular Genes ◽  
Long Read ◽  
Drosophila Brain ◽  
Cryptic Splice Sites ◽  
Somatic Transposition

AbstractSomatic transposition in neural tissue could contribute to neuropathology and individuality, but its prevalence is debated. We used single-cell mRNA sequencing to map transposon expression in the Drosophila midbrain. We found that neural transposon expression is driven by cellular genes. Every expressed transposon is resident in at least one cellular gene with a matching expression pattern. A new long-read RNA sequencing approach revealed that coexpression is a physical link in the form of abundant chimeric transposon-gene mRNAs. We identified 148 genes where transposons introduce cryptic splice sites into the nascent transcript and thereby produce many additional mRNAs. Some genes exclusively produce chimeric mRNAs with transposon sequence and on average transposon-gene chimeras account for 20% of the mRNAs produced from a given gene. Transposons therefore significantly expand the neural transcriptome. We propose that chimeric mRNAs produced by splicing into polymorphic transposons may contribute to functional differences between individual cells and animals.

scholarly journals Yeast and Human RNA Polymerase II Elongation Complexes: Evidence for Functional Differences and Postinitiation Recruitment of Factors

2003 ◽  
Vol 2 (2) ◽  
pp. 318-327 ◽  
Author(s):  
Timothy S. Pardee ◽  
Mohamed A. Ghazy ◽  
Alfred S. Ponticelli
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Exonuclease Activity ◽  
Nascent Transcript ◽  
Dna Templates ◽  
Functional Differences ◽  
Nuclear Extracts ◽  
Nuclear Factors

ABSTRACT Immobilized DNA templates, glycerol gradient centrifugation, and native gel analysis were utilized to isolate and compare functional RNA polymerase II (RNAPII) elongation complexes from Saccharomyces cerevisiae and human cell nuclear extracts. Yeast elongation complexes blocked by incorporation of 3′-O-methyl-GTP into the nascent transcript exhibited a sedimentation coefficient of 35S, were less tightly associated to the template than their human counterparts, and displayed no detectable 3′-5′ exonuclease activity on the associated transcript. In contrast, blocked human elongation complexes were more tightly bound to the template, and multiple forms were identified, with the largest exhibiting a sedimentation coefficient of 60S. Analysis of the associated transcripts revealed that a subset of the human elongation complexes exhibited strong 3′-5′ exonuclease activity. Although isolated human preinitiation complexes were competent for efficient transcription, their ability to generate 60S elongation complexes was strikingly impaired. These findings demonstrate functional and size differences between S. cerevisiae and human RNAPII elongation complexes and support the view that the formation of mature elongation complexes involves recruitment of nuclear factors after the initiation of transcription.

scholarly journals Precise gene models using long-read sequencing reveal a unique poly(A) signal in Giardia lamblia

2021 ◽  
Author(s):  
Danielle Y Bilodeau ◽  
Ryan M Sheridan ◽  
Balu Balan ◽  
Aaron R Jex ◽  
Olivia S Rissland
Keyword(s):  
Gene Expression ◽  
Protein Complex ◽  
Giardia Lamblia ◽  
Alternative Polyadenylation ◽  
Mrna Processing ◽  
Cleavage Sites ◽  
Nascent Transcript ◽  
Long Read ◽  
Show Evidence ◽  
Cleavage And Polyadenylation

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Here, using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses a AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes that show evidence of alternative polyadenylation. These results suggest that despite pared down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in G. lamblia.

scholarly journals Inversions maintain differences between migratory phenotypes of a songbird

2021 ◽  
Author(s):  
Max Lundberg ◽  
Alexander Mackintosh ◽  
Anna Petri ◽  
Staffan Bensch
Keyword(s):  
Environmental Gradients ◽  
Segmental Duplications ◽  
Migration Routes ◽  
Long Distance ◽  
Local Adaptations ◽  
Functional Differences ◽  
Long Read ◽  
Wintering Areas ◽  
Genome Assemblies ◽  
Chromosome Regions

Long-distance migration requires adaptations in a suite of behavioral, physiological and morphological traits. However, almost nothing is known about the genetic basis of these adaptations. The willow warbler Phylloscopus trochilus occurs in Europe with a northern and southern subspecies that show drastically different migration routes and wintering areas. Previous studies have demonstrated that the subspecies are genetically extremely similar except for three divergent chromosome regions, of which two are associated with the differences in migratory phenotypes and one is associated with an environmental gradient. Here we use a combination of long-read sequencing, linked-read sequencing and optical mapping to construct more complete and contiguous assemblies for both of the subspecies. We find evidence for inversions in each of the three divergent regions, which range from 0.4 to 13 Mb in size, and that breakpoints are associated with tandem repeat arrays or segmental duplications. The divergence times between inverted and non-inverted haplotypes are similar across the regions (~1.2 Myrs), which is compatible with a scenario where the inversions arose in either of two allopatric populations that subsequently hybridized. The improved genome assemblies and annotation also allowed us to detect additional functional differences in the divergent regions that provide candidate genes for migration and local adaptations to environmental gradients.

scholarly journals Long-read sequencing of nascent RNA reveals coupling among RNA processing events

2017 ◽  
Author(s):  
Lydia Herzel ◽  
Korinna Straube ◽  
Karla M. Neugebauer
Keyword(s):  
De Novo ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
Nascent Transcript ◽  
Long Read ◽  
Constitutive Splicing ◽  
Nuclear Exosome ◽  
Nascent Transcripts ◽  
Mrna Half Life ◽  
Regulatory Significance

AbstractPre-mRNA splicing is accomplished by the spliceosome, a megadalton complex that assembles de novo on each intron. Because spliceosome assembly and catalysis occur co-transcriptionally, we hypothesized that introns are removed in the order of their transcription in genomes dominated by constitutive splicing. Remarkably little is known about splicing order and the regulatory potential of nascent transcript remodeling by splicing, due to the limitations of existing methods that focus on analysis of mature splicing products (mRNAs) rather than substrates and intermediates. Here, we overcome this obstacle through long-read RNA sequencing of nascent, multi-intron transcripts in the fission yeast Schizosaccharomyces pombe. Most multi-intron transcripts were fully spliced, consistent with rapid co-transcriptional splicing. However, an unexpectedly high proportion of transcripts were either fully spliced or fully unspliced, suggesting that splicing of any given intron is dependent on the splicing status of other introns in the transcript. Supporting this, mild inhibition of splicing by a temperature-sensitive mutation in Prp2, the homolog of vertebrate U2AF65, increased the frequency of fully unspliced transcripts. Importantly, fully unspliced transcripts displayed transcriptional read-through at the polyA site and were degraded co-transcriptionally by the nuclear exosome. Finally, we show that cellular mRNA levels were reduced in genes with a high number of unspliced nascent transcripts during caffeine treatment, showing regulatory significance of co-transcriptional splicing. Therefore, overall splicing of individual nascent transcripts, 3’ end formation, and mRNA half-life depend on the splicing status of neighboring introns, suggesting crosstalk among spliceosomes and the polyA cleavage machinery during transcription elongation.

scholarly journals Control of Rta expression critically determines transcription of viral and cellular genes following gammaherpesvirus infection

2007 ◽  
Vol 88 (6) ◽  
pp. 1689-1697 ◽  
Author(s):  
James R. Hair ◽  
Paul A. Lyons ◽  
Kenneth G. C. Smith ◽  
Stacey Efstathiou
Keyword(s):  
Gene Expression ◽  
Critical Role ◽  
Lytic Cycle ◽  
Productive Infection ◽  
Cellular Gene ◽  
Cellular Gene Expression ◽  
Murine Gammaherpesvirus ◽  
Cellular Genes ◽  
The Impact

The replication and transcriptional activator (Rta), encoded by ORF50 of gammaherpesviruses, initiates the lytic cycle of gene expression; therefore understanding the impact of Rta on viral and cellular gene expression is key to elucidating the transcriptional events governing productive infection and reactivation from latency. To this end, the impact of altering Rta transcription on viral and cellular gene expression was studied in the context of a whole virus infection. Recombinant murine gammaherpesvirus (MHV)-68 engineered to overexpress Rta greatly accelerated expression of specific lytic cycle ORFs, but repressed transcription of the major latency gene, ORF73. Increased expression of Rta accelerated the dysregulation in transcription of specific cellular genes when compared with cells infected with wild-type and revertant viruses. A subset of cellular genes was dysregulated only in cells infected with Rta-overexpressing virus, and never in those infected with non-overexpressing viruses. These data highlight the critical role of Rta abundance in governing viral and cellular gene transcription, and demonstrate the importance of understanding how the relative expression of ORF50 during the virus life cycle impacts on these processes.

The cHS4 Chromatin Insulator Reduces the Rate of Retroviral Vector-Mediated Gene Dysregulation Associated with Aberrant Vector Transcription

2017 ◽  
Vol 151 (2) ◽  
pp. 72-81
Author(s):  
Xianyao Zhou ◽  
Qiujun Liu ◽  
Da Wang ◽  
Xuemei Zhang ◽  
David W. Emery ◽  
...  
Keyword(s):  
Malignant Transformation ◽  
Transcription Termination ◽  
Retroviral Vector ◽  
Gene Sequences ◽  
Cellular Gene ◽  
Aberrant Splicing ◽  
Chromatin Insulator ◽  
Fusion Transcripts ◽  
Gene Dysregulation ◽  
Cellular Genes

Integrating gammaretroviral vectors can dysregulate the expression of cellular genes through a variety of mechanisms, leading to genotoxicity and malignant transformation. Although most attention has focused on the activation of cellular genes by vector enhancers, aberrant fusion transcripts involving cellular gene sequences and vector promoters, vector splice elements, and vector transcription termination sequences have also been mechanistically associated with dysregulated expression of cellular genes. Chromatin insulators have emerged as an effective tool for reducing the frequency of vector-mediated genotoxicity and malignant transformation and have been shown to block the activation of cellular genes by vector enhancers. We report here evidence that flanking a gammaretroviral reporter vector with the cHS4 chromatin insulator also reduces the frequency of vector-mediated cellular gene dysregulation associated with aberrant vector transcripts, including vector transcription run-through and aberrant splicing. We demonstrate that the cHS4 element does not function to terminate transcription directly, implicating other mechanisms for this activity.

scholarly journals EBNA2 and Activated Notch Induce Expression of BATF

2003 ◽  
Vol 77 (10) ◽  
pp. 6029-6040 ◽  
Author(s):  
Lisa M. Johansen ◽  
Christopher D. Deppmann ◽  
Kimberly D. Erickson ◽  
William F. Coffin ◽  
Tina M. Thornton ◽  
...  
Keyword(s):  
B Cells ◽  
Target Genes ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Negative Regulator ◽  
Cellular Gene ◽  
Barr Virus ◽  
Cellular Genes ◽  
Latently Infected ◽  
Epstein Barr

ABSTRACT The immortalization of human B lymphocytes by Epstein-Barr virus (EBV) requires the virus-encoded transactivator EBNA2 and the products of both viral and cellular genes which serve as EBNA2 targets. In this study, we identified BATF as a cellular gene that is up-regulated dramatically within 24 h following the infection of established and primary human B cells with EBV. The transactivation of BATF is mediated by EBNA2 in a B-cell-specific manner and is duplicated in non-EBV-infected B cells by the expression of mammalian Notch proteins. In contrast to other target genes activated by EBNA2, the BATF gene encodes a member of the AP-1 family of transcription factors that functions as a negative regulator of AP-1 activity and as an antagonist of cell growth. A potential role for BATF in promoting EBV latency is supported by studies in which BATF was shown to negatively impact the expression of a BZLF1 reporter gene and to reduce the frequency of lytic replication in latently infected cells. The identification of BATF as a cellular target of EBV provides important new information on how programs of viral and cellular gene expression may be coordinated to promote viral latency and control lytic-cycle entry.

scholarly journals Full-length poly(A) and mRNA sequencing (FLAM-seq)

2019 ◽  
Author(s):  
Ivano Legnini ◽  
Jonathan Alles ◽  
Nikos Karaiskos ◽  
Salah Ayoub ◽  
Claudia Quedenau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tail Length ◽  
Full Length ◽  
Cdna Libraries ◽  
Simple Method ◽  
Mrna Sequencing ◽  
New Strategy ◽  
Long Read ◽  
Mrna Isoform ◽  
Cdna Preparation

Abstract We developed FLAM-seq, a fast and simple method for generating cDNA libraries of full-length mRNAs, including the poly(A) tail. By combining a new strategy for cDNA preparation with PacBio long-read sequencing, FLAM-seq enables to generate hundreds of thousands of reads per sample in an easy and short procedure, with starting material ranging from 500 ng to 10 μg of total RNA. Besides the quantification of gene expression and the information about the mRNA isoform, the data generated with FLAM-seq allow accurate measurement of poly(A) tail length for thousands of genes in the sequenced samples. Here we describe the protocol step by step from the RNA preparation to the first steps of data analysis.

scholarly journals LongGF: computational algorithm and software tool for fast and accurate detection of gene fusions by long-read transcriptome sequencing

BMC Genomics ◽  
2020 ◽  
Vol 21 (S11) ◽  
Author(s):  
Qian Liu ◽  
Yu Hu ◽  
Andres Stucky ◽  
Li Fang ◽  
Jiang F. Zhong ◽  
...  
Keyword(s):  
Candidate Gene ◽  
Gene Fusion ◽  
Superior Performance ◽  
Gene Fusions ◽  
Rna Seq ◽  
Cdna Sequencing ◽  
Sequencing Data ◽  
Mrna Sequencing ◽  
Long Read ◽  
Fusion Detection

Abstract Background Long-read RNA-Seq techniques can generate reads that encompass a large proportion or the entire mRNA/cDNA molecules, so they are expected to address inherited limitations of short-read RNA-Seq techniques that typically generate < 150 bp reads. However, there is a general lack of software tools for gene fusion detection from long-read RNA-seq data, which takes into account the high basecalling error rates and the presence of alignment errors. Results In this study, we developed a fast computational tool, LongGF, to efficiently detect candidate gene fusions from long-read RNA-seq data, including cDNA sequencing data and direct mRNA sequencing data. We evaluated LongGF on tens of simulated long-read RNA-seq datasets, and demonstrated its superior performance in gene fusion detection. We also tested LongGF on a Nanopore direct mRNA sequencing dataset and a PacBio sequencing dataset generated on a mixture of 10 cancer cell lines, and found that LongGF achieved better performance to detect known gene fusions over existing computational tools. Furthermore, we tested LongGF on a Nanopore cDNA sequencing dataset on acute myeloid leukemia, and pinpointed the exact location of a translocation (previously known in cytogenetic resolution) in base resolution, which was further validated by Sanger sequencing. Conclusions In summary, LongGF will greatly facilitate the discovery of candidate gene fusion events from long-read RNA-Seq data, especially in cancer samples. LongGF is implemented in C++ and is available at https://github.com/WGLab/LongGF.

scholarly journals The Adenoviral E1B 55-Kilodalton Protein Controls Expression of Immune Response Genes but Not p53-Dependent Transcription

2009 ◽  
Vol 83 (8) ◽  
pp. 3591-3603 ◽  
Author(s):  
Daniel L. Miller ◽  
Brenden Rickards ◽  
Michael Mashiba ◽  
Wenying Huang ◽  
S. J. Flint
Keyword(s):  
Gene Expression ◽  
Human Fibroblasts ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Cellular Gene ◽  
Cellular Gene Expression ◽  
Cellular Processes ◽  
Hybridization Data ◽  
Cellular Genes ◽  
Genes Encoding

ABSTRACT The human adenovirus type 5 (Ad5) E1B 55-kDa protein modulates several cellular processes, including activation of the tumor suppressor p53. Binding of the E1B protein to the activation domain of p53 inhibits p53-dependent transcription. This activity has been correlated with the transforming activity of the E1B protein, but its contribution to viral replication is not well understood. To address this issue, we used microarray hybridization methods to examine cellular gene expression in normal human fibroblasts (HFFs) infected by Ad5, the E1B 55-kDa-protein-null mutant Hr6, or a mutant carrying substitutions that impair repression of p53-dependent transcription. Comparison of the changes in cellular gene expression observed in these and our previous experiments (D. L. Miller et al., Genome Biol. 8:R58, 2007) by significance analysis of microarrays indicated excellent reproducibility. Furthermore, we again observed that Ad5 infection led to efficient reversal of the p53-dependent transcriptional program. As this same response was also induced in cells infected by the two mutants, we conclude that the E1B 55-kDa protein is not necessary to block activation of p53 in Ad5-infected cells. However, groups of cellular genes that were altered in expression specifically in the absence of the E1B protein were identified by consensus k-means clustering of the hybridization data. Statistical analysis of the enrichment of genes associated with specific functions in these clusters established that the E1B 55-kDa protein is necessary for repression of genes encoding proteins that mediate antiviral and immune defenses.

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