-
PLoS Genetics May 2024Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance...
Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance patterns deviating from Mendelian expectations in a behavior known as paramutation. However, the chromosome features mediating such exceptions remain unknown. Here we show that small RNA production reflecting RNA polymerase IV function within a distal downstream set of five tandem repeats is coincident with meiotically-heritable repression of the Pl1-Rhoades transcription unit. A related pl1 haplotype with three, but not one with two, repeat units also displays the trans-homolog silencing typifying paramutations. 4C interactions, CHD3a-dependent small RNA profiles, nuclease sensitivity, and polyadenylated RNA levels highlight a repeat subregion having regulatory potential. Our comparative and mutant analyses show that transcriptional repression of Pl1-Rhoades correlates with 24-nucleotide RNA production and cytosine methylation at this subregion indicating the action of a specific DNA-dependent RNA polymerase complex. These findings support a working model in which pl1 paramutation depends on trans-chromosomal RNA-directed DNA methylation operating at a discrete cis-linked and copy-number-dependent transcriptional regulatory element.
PubMed: 38814980
DOI: 10.1371/journal.pgen.1011296 -
BMC Genomics May 2024Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing...
BACKGROUND
Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been expanding the potential of dRNA-seq, its sequencing accuracy and error patterns remain understudied.
RESULTS
We present the first comprehensive evaluation of sequencing accuracy and characterisation of systematic errors in dRNA-seq data from diverse organisms and synthetic in vitro transcribed RNAs. We found that for sequencing kits SQK-RNA001 and SQK-RNA002, the median read accuracy ranged from 87% to 92% across species, and deletions significantly outnumbered mismatches and insertions. Due to their high abundance in the transcriptome, heteropolymers and short homopolymers were the major contributors to the overall sequencing errors. We also observed systematic biases across all species at the levels of single nucleotides and motifs. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines and adenines. By examining raw signal data, we identified the underlying signal-level features potentially associated with the error patterns and their dependency on sequence contexts. While read quality scores can be used to approximate error rates at base and read levels, failure to detect DNA adapters may be a source of errors and data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (basecalling) artefacts. Lastly, we generated dRNA-seq data using the latest SQK-RNA004 sequencing kit released at the end of 2023 and found that although the overall read accuracy increased, the systematic errors remain largely identical compared to the previous kits.
CONCLUSIONS
As the first systematic investigation of dRNA-seq errors, this study offers a comprehensive overview of reproducible error patterns across diverse datasets, identifies potential signal-level insufficiency, and lays the foundation for error correction methods.
Topics: Sequence Analysis, RNA; Nanopore Sequencing; Nanopores; Humans; Animals; RNA; High-Throughput Nucleotide Sequencing
PubMed: 38807060
DOI: 10.1186/s12864-024-10440-w -
Data in Brief Jun 2024is a widespread shell-less species of nudibranch molluscs, which has unique for Gastropoda skeletal elements - subepidermal calcite spicules. The general and fine...
is a widespread shell-less species of nudibranch molluscs, which has unique for Gastropoda skeletal elements - subepidermal calcite spicules. The general and fine morphology of the spicules, as well as their maturation process in ontogenesis, have been studied in detail by authors. The uniqueness of spicules lies in their intracellular formation and location under the ectodermal epithelium, which is more typical for deuterostomes. We present as a potentially new model species for studying calcification of intracellular protein structure. A total of 96 individuals were collected in the Kandalaksha Bay of the White Sea, both manually and by scuba diving. All individuals were divided into three groups based on morphological characteristics such as specimens' size, spicule condition etc. This division suggests the existence of three stages in postembryonic ontogenesis of reflecting the maturation of the spicule complex. Total RNA samples were isolated from three size groups of molluscs in three biological replicates. Libraries were prepared from the polyadenylated RNA fraction and sequenced at NovaSeq6000 (Illumina), yielding a total of 112.8 Gb of 150 bp paired-end reads, corresponding to almost 1,000-fold coverage of the transcriptome. Representative transcriptome assembled with Trinity. In addition to obtaining the transcriptome sequences of , differential expression analysis was also performed for these three size groups. This allows us to trace the dynamics of molecular and biological processes during the life of a mollusc. The obtained data can then be used as a reference transcriptome for closely related species, to study specific expressed genes, to identify various unique sequences, including protein-coding ones, to understand biological processes, including biomineralization and much more.
PubMed: 38799714
DOI: 10.1016/j.dib.2024.110526 -
RNA Biology Jan 2024Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant...
Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant challenge to characterize them from a biological sample. In this study, we assessed the outcomes of bovine circRNA identification using six enrichment approaches with the combination of ribosomal RNAs removal (); linear RNAs degradation (); linear RNAs and RNAs with structured 3' ends degradation (); ribosomal RNAs coupled with linear RNAs elimination (); ribosomal RNA, linear RNAs and RNAs with poly (A) tailing elimination (); and ribosomal RNA, linear RNAs and RNAs with structured 3' ends elimination (), respectively. RNA-sequencing analysis revealed that different approaches led to varied ratio of uniquely mapped reads, false-positive rate of identifying circRNAs, and the number of circRNAs per million clean reads ( <0.05). Out of 2,285 and 2,939 highly confident circRNAs identified in liver and rumen tissues, respectively, 308 and 260 were commonly identified from five methods, with Ribo-RTP method identified the highest number of circRNAs. Besides, 507 of 4,051 identified bovine highly confident circRNAs had shared splicing sites with human circRNAs. The findings from this work provide optimized methods to identify bovine circRNAs from cattle tissues for downstream research of their biological roles in cattle.
Topics: Cattle; RNA, Circular; Animals; RNA, Ribosomal; Sequence Analysis, RNA; Liver; Rumen; Computational Biology; Gene Expression Profiling; Humans
PubMed: 38797889
DOI: 10.1080/15476286.2024.2356334 -
Viruses May 2024Papillomavirus gene regulation is largely post-transcriptional due to overlapping open reading frames and the use of alternative polyadenylation and alternative splicing... (Review)
Review
Papillomavirus gene regulation is largely post-transcriptional due to overlapping open reading frames and the use of alternative polyadenylation and alternative splicing to produce the full suite of viral mRNAs. These processes are controlled by a wide range of cellular RNA binding proteins (RPBs), including constitutive splicing factors and cleavage and polyadenylation machinery, but also factors that regulate these processes, for example, SR and hnRNP proteins. Like cellular RNAs, papillomavirus RNAs have been shown to bind many such proteins. The life cycle of papillomaviruses is intimately linked to differentiation of the epithelial tissues the virus infects. For example, viral late mRNAs and proteins are expressed only in the most differentiated epithelial layers to avoid recognition by the host immune response. Papillomavirus genome replication is linked to the DNA damage response and viral chromatin conformation, processes which also link to RNA processing. Challenges with respect to elucidating how RBPs regulate the viral life cycle include consideration of the orchestrated spatial aspect of viral gene expression in an infected epithelium and the epigenetic nature of the viral episomal genome. This review discusses RBPs that control viral gene expression, and how the connectivity of various nuclear processes might contribute to viral mRNA production.
Topics: RNA-Binding Proteins; Humans; Gene Expression Regulation, Viral; RNA, Viral; Papillomaviridae; Virus Replication; Viral Proteins; Papillomavirus Infections; Genome, Viral; Host-Pathogen Interactions; RNA, Messenger
PubMed: 38793664
DOI: 10.3390/v16050783 -
Viruses Apr 2024The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including...
The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including CPSF6, that direct HIV-1 integration into speckle-associated domains in host chromatin. Upon HIV-1 infection, CPSF6 forms puncta in the nucleus. Here, we characterised these CPSF6 puncta further in HeLa cells, T-cells and macrophages and confirmed that integration and reverse transcription are not required for puncta formation. Indeed, we found that puncta formed very rapidly after infection, correlating with the time that CA entered the nucleus. In aphidicolin-treated HeLa cells and macrophages, puncta were detected for the length of the experiment, suggesting that puncta are only lost upon cell division. CA still co-localised with CPSF6 puncta at the latest time points, considerably after the peak of reverse transcription and integration. Intriguingly, the number of puncta induced in macrophages did not correlate with the MOI or the total number of nuclear speckles present in each cell, suggesting that CA/CPSF6 is only directed to a few nuclear speckles. Furthermore, we found that CPSF6 already co-localised with nuclear speckles in uninfected T-cells, suggesting that HIV-1 promotes a natural behaviour of CPSF6.
Topics: HIV-1; Humans; mRNA Cleavage and Polyadenylation Factors; T-Lymphocytes; HeLa Cells; Macrophages; Virus Integration; Cell Nucleus; Capsid Proteins; HIV Infections; Capsid
PubMed: 38793552
DOI: 10.3390/v16050670 -
NPJ Parkinson's Disease May 2024A biallelic (AAGGG) expansion in the poly(A) tail of an AluSx3 transposable element within the gene RFC1 is a frequent cause of cerebellar ataxia, neuropathy, vestibular...
A biallelic (AAGGG) expansion in the poly(A) tail of an AluSx3 transposable element within the gene RFC1 is a frequent cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS), and more recently, has been reported as a rare cause of Parkinson's disease (PD) in the Finnish population. Here, we investigate the prevalence of RFC1 (AAGGG) expansions in PD patients of non-Finnish European ancestry in 1609 individuals from the Parkinson's Progression Markers Initiative study. We identified four PD patients carrying the biallelic RFC1 (AAGGG) expansion and did not identify any carriers in controls.
PubMed: 38789445
DOI: 10.1038/s41531-024-00723-0 -
Cells May 2024Mammalian oocyte development depends on the temporally controlled translation of maternal transcripts, particularly in the coordination of meiotic and early embryonic...
Mammalian oocyte development depends on the temporally controlled translation of maternal transcripts, particularly in the coordination of meiotic and early embryonic development when transcription has ceased. The translation of mRNA is regulated by various RNA-binding proteins. We show that the absence of cytoplasmic polyadenylation element-binding protein 3 (CPEB3) negatively affects female reproductive fitness. CPEB3-depleted oocytes undergo meiosis normally but experience early embryonic arrest due to a disrupted transcriptome, leading to aberrant protein expression and the subsequent failure of embryonic transcription initiation. We found that CPEB3 stabilizes a subset of mRNAs with a significantly longer 3'UTR that is enriched in its distal region with cytoplasmic polyadenylation elements. Overall, our results suggest that CPEB3 is an important maternal factor that regulates the stability and translation of a subclass of mRNAs that are essential for the initiation of embryonic transcription and thus for embryonic development.
Topics: Oocytes; Animals; RNA-Binding Proteins; Female; Mice; Meiosis; RNA, Messenger; Embryonic Development; Gene Expression Regulation, Developmental; 3' Untranslated Regions; Polyadenylation; RNA Stability
PubMed: 38786074
DOI: 10.3390/cells13100850 -
Microbial Cell (Graz, Austria) 2024In , polyadenylated forms of mature (and not precursor) small non-coding RNAs (sncRNAs) those fail to undergo proper 3'-end maturation are subject to an active...
In , polyadenylated forms of mature (and not precursor) small non-coding RNAs (sncRNAs) those fail to undergo proper 3'-end maturation are subject to an active degradation by Rrp6p and Rrp47p, which does not require the involvement of core exosome and TRAMP components. In agreement with this finding, Rrp6p/Rrp47p is demonstrated to exist as an exosome-independent complex, which preferentially associates with mature polyadenylated forms of these sncRNAs. Consistent with this observation, a C-terminally truncated version of Rrp6p (Rrp6p-ΔC2) lacking physical association with the core nuclear exosome supports their decay just like its full-length version. Polyadenylation is catalyzed by both the canonical and non-canonical poly(A) polymerases, Pap1p and Trf4p. Analysis of the polyadenylation profiles in WT and -Δ strains revealed that the majority of the polyadenylation sites correspond to either one to three nucleotides upstream or downstream of their mature ends and their poly(A) tails ranges from 10-15 adenylate residues. Most interestingly, the accumulated polyadenylated snRNAs are functional in the -Δ strain and are assembled into spliceosomes. Thus, Rrp6p-Rrp47p defines a core nuclear exosome-independent novel RNA turnover system in baker's yeast targeting imperfectly processed polyadenylated sncRNAs that accumulate in the absence of Rrp6p.
PubMed: 38783922
DOI: 10.15698/mic2024.05.823 -
DIPAN: Detecting personalized intronic polyadenylation derived neoantigens from RNA sequencing data.Computational and Structural... Dec 2024Intronic polyadenylation (IPA) refers to a particular type of alternative polyadenylation where a gene makes use of a polyadenylation site located within its introns....
Intronic polyadenylation (IPA) refers to a particular type of alternative polyadenylation where a gene makes use of a polyadenylation site located within its introns. Aberrant IPA events have been observed in various types of cancer. IPA can produce noncoding transcripts or truncated protein-coding transcripts with altered coding sequences in the resulting protein product. Therefore, IPA events hold the potential to act as a reservoir of tumor neoantigens. Here, we developed a computational method termed DIPAN, which incorporates IPA detection, protein fragmentation, and MHC binding prediction to predict IPA-derived neoantigens. Utilizing RNA-seq from breast cancer cell lines and ovarian cancer clinical samples, we demonstrated the significant contribution of IPA events to the neoantigen repertoire. Through mass spectrometry immunopeptidome analysis, we further illustrated the processing and presentation of IPA-derived neoantigens on the surface of cancer cells. While most IPA-derived neoantigens are sample-specific, shared neoantigens were identified in both cancer cell lines and clinical samples. Furthermore, we demonstrated an association between IPA-derived neoantigen burden and overall survival in cancer patients.
PubMed: 38783901
DOI: 10.1016/j.csbj.2024.05.008