Alternative Transcription Termination Research Articles

Identification of antennal alternative splicing by combining genome and full-length transcriptome analysis in Bactrocera dorsalis.

Alternative splicing is an essential post-transcriptional regulatory mechanism that diversifies gene function by generating multiple protein isoforms from a single gene and act as a crucial role in insect environmental adaptation. Olfaction, a key sense for insect adaptation, relies heavily on the antennae, which are the primary olfactory organs expressing most of the olfactory genes. Despite the extensive annotation of olfactory genes within insect antennal tissues facilitated by high-throughput sequencing technology advancements, systematic analyses of alternative splicing are still relatively less. In this study, we focused on the oriental fruit fly (Bactrocera dorsalis), a significant pest of fruit crops. We performed a detailed analysis of alternative splicing in its antennae by utilizing the full-length transcriptome of its antennal tissue and the insect's genome. The results revealed 8600 non-redundant full-length transcripts identified in the oriental fruit fly antennal full-length transcriptome, spanning 4,145 gene loci. Over 40% of these loci exhibited multiple isoforms. Among these, 161 genes showed sex-biased isoform switching, involving seven different types of alternative splicing. Notably, events involving alternative transcription start sites (ATSS) and alternative transcription termination sites (ATTS) were the most common. Of all the genes undergoing ATSS and ATTS alternative splicing between male and female, 32 genes were alternatively spliced in protein coding regions, potentially affecting protein function. These genes were categorized based on the length of the sex-biased isoforms, with the highest difference in isoform fraction (dIF) associated with the ATSS type, including genes such as BdorABCA13, BdorCAT2, and BdorTSN3. Additionally, transcription factor binding sites for doublesex were identified upstream of both BdorABCA13 and BdorCAT2. Besides being expressed in the antennal tissues, BdorABCA13 and BdorCAT2 are also expressed in the mouthparts, legs, and genitalia of both female and male adults, suggesting their functional diversity. This study reveals alternative splicing events in the antennae of Bactrophora dorsalis from two aspects: odorant receptor genes and other types of genes expressed in the antennae. This study not only provides a research foundation for understanding the regulation of gene function by alternative splicing in the oriental fruit fly but also offers new insights for utilizing olfaction-based behavioral manipulation techniques to manage this pest.

Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination

Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.

Splicing Characterization and Isoform Switch Events in Human Keratinocytes Carrying Oncogenes from High-Risk HPV-16 and Low-Risk HPV-84.

Infection of epithelial cells with high-risk HPV (HR-HPV) types, followed by expression of virus oncogenic proteins (E5, E6, and E7), leads to genomic imbalance, suppression of tumor inhibitors, and induction of oncogenes. Low-risk HPV (LR-HPV) may slow the rate at which cervical cancer spreads to an invasive stage since co-infection with LR-HPV is linked to a decreased risk of future invasive cancer than infection with HR-HPV alone. We then propose that cancer-progressing changes may be distinguished through identifying the functional differences between LR-HPV and HR-HPV. Lentiviral strategies were followed to establish HaCaT cells with constitutive expression of HPV oncogenes. RNAseq experiments were designed to analyze the transcriptome modulations caused by each of the E5, E6, and E7 oncogenes of HPV-16 and HPV-84 in HaCaT cells. We identified enhanced RNA degradation, spliceosome, and RNA polymerase pathways related to mRNA processing. ATTS (alternative transcription termination site) was discovered to be more prevalent in cells with HPV-16E5 than HPV-84E5. In HPV-16E6-infected cells, ATTS gain was significantly higher than ATTS loss. Cells with HPV-16E7 had more isoforms with intron retention (IR) than those with HPV-84E7. We identified switches in ADAM10, CLSPN, and RNPS1 that led to greater expression of the coding isoforms in HR-HPV. The results of this work highlight differences between LR-HPV and HR-HPV in mRNA processing. Moreover, crucial cervical cancer-related switch events were detected.

Functional dissection of NEAT1 using genome editing reveals substantial localization of the NEAT1_1 isoform outside paraspeckles.

Large numbers of long noncoding RNAs have been discovered in recent years, but only a few have been characterized. NEAT1 (nuclear paraspeckle assembly transcript 1) is a mammalian long noncoding RNA that is important for the reproductive physiology of mice, cancer development, and the formation of subnuclear bodies termed paraspeckles. The two major isoforms of NEAT1 (3.7 kb NEAT1_1 and 23 kb NEAT1_2 in human) are generated from a common promoter and are produced through the use of alternative transcription termination sites. This gene structure has made the functional relationship between the two isoforms difficult to dissect. Here we used CRISPR-Cas9 genome editing to create several different cell lines: total NEAT1 knockout cells, cells that only express the short form NEAT1_1, and cells with twofold more NEAT1_2. Using these reagents, we obtained evidence that NEAT1_1 is not a major component of paraspeckles. In addition, our data suggest NEAT1_1 localizes in numerous nonparaspeckle foci we termed “microspeckles,” which may carry paraspeckle-independent functions. This study highlights the complexity of lncRNA and showcases how genome editing tools are useful in dissecting the structural and functional roles of overlapping transcripts.

Mechanism of alternative splicing and its regulation.

Alternative splicing of precursor mRNA is an essential mechanism to increase the complexity of gene expression, and it plays an important role in cellular differentiation and organism development. Regulation of alternative splicing is a complicated process in which numerous interacting components are at work, including cis-acting elements and trans-acting factors, and is further guided by the functional coupling between transcription and splicing. Additional molecular features, such as chromatin structure, RNA structure and alternative transcription initiation or alternative transcription termination, collaborate with these basic components to generate the protein diversity due to alternative splicing. All these factors contributing to this one fundamental biological process add up to a mechanism that is critical to the proper functioning of cells. Any corruption of the process may lead to disruption of normal cellular function and the eventuality of disease. Cancer is one of those diseases, where alternative splicing may be the basis for the identification of novel diagnostic and prognostic biomarkers, as well as new strategies for therapy. Thus, an in-depth understanding of alternative splicing regulation has the potential not only to elucidate fundamental biological principles, but to provide solutions for various diseases.

Evolution at protein ends: major contribution of alternative transcription initiation and termination to the transcriptome and proteome diversity in mammals.

Alternative splicing (AS), alternative transcription initiation (ATI) and alternative transcription termination (ATT) create the extraordinary complexity of transcriptomes and make key contributions to the structural and functional diversity of mammalian proteomes. Analysis of mammalian genomic and transcriptomic data shows that contrary to the traditional view, the joint contribution of ATI and ATT to the transcriptome and proteome diversity is quantitatively greater than the contribution of AS. Although the mean numbers of protein-coding constitutive and alternative nucleotides in gene loci are nearly identical, their distribution along the transcripts is highly non-uniform. On average, coding exons in the variable 5′ and 3′ transcript ends that are created by ATI and ATT contain approximately four times more alternative nucleotides than core protein-coding regions that diversify exclusively via AS. Short upstream exons that encompass alternative 5′-untranslated regions and N-termini of proteins evolve under strong nucleotide-level selection whereas in 3′-terminal exons that encode protein C-termini, protein-level selection is significantly stronger. The groups of genes that are subject to ATI and ATT show major differences in biological roles, expression and selection patterns.

Connections between Alternative Transcription and Alternative Splicing in Mammals

The majority of mammalian genes produce multiple transcripts resulting from alternative splicing (AS) and/or alternative transcription initiation (ATI) and alternative transcription termination (ATT). Comparative analysis of the number of alternative nucleotides, isoforms, and introns per locus in genes with different types of alternative events suggests that ATI and ATT contribute to the diversity of human and mouse transcriptome even more than AS. There is a strong negative correlation between AS and ATI in 5′ untranslated regions (UTRs) and AS in coding sequences (CDSs) but an even stronger positive correlation between AS in CDSs and ATT in 3′ UTRs. These observations could reflect preferential regulation of distinct, large groups of genes by different mechanisms: 1) regulation at the level of transcription initiation and initiation of translation resulting from ATI and AS in 5′ UTRs and 2) posttranslational regulation by different protein isoforms. The tight linkage between AS in CDSs and ATT in 3′ UTRs suggests that variability of 3′ UTRs mediates differential translational regulation of alternative protein forms. Together, the results imply coordinate evolution of AS and alternative transcription, processes that occur concomitantly within gene expression factories.

Transcription of the German cockroach densovirus BgDNV genome: Alternative processing of viral RNAs

176 Parvoviruses are among the smallest DNA animal viruses: their single-stranded genome is 4‐6 thousand nucleotides long. For this reason, alternative splicing of viral pre-mRNAs plays a major role in increasing the coding ability of their small genome. In all studied vertebrate parvoviruses, expression is regulated primarily at the transcriptional level. However, transcription and processing of RNAs of the majority of densoviruses has not yet been studied. In this work, we studied the consistent transcription patterns of the German cockroach densovirus BgDNV. This study provided new information on the expression of this densovirus, which has not been described for any other insect densovirus. These data are important not only for studying particular characteristics of the life cycle of viruses but also for understanding the fundamentals of regulation of eukaryotic gene expression. When BGE2 cockroach cell culture is infected with the BgDNV virus, several viral RNAs, formed as a result of alternative splicing and use of alternative transcription termination codons, are transcribed from each DNA strand of the viral genome. It was shown that splicing of RNAs encoding both VP and NS proteins is observed during infection development and that the viral RNAs for NS proteins undergo alternative splicing. We determined the size of the transcripts synthesized from two viral promoters and showed that the transcription from the NS promoter starts much earlier than the transcription from the VP promoter. We also showed that the transcripts synthesized from antiparallel DNA strands of the BgDNV virus genome considerably overlap as a result of RNA processing.

Cloning and transcription analysis of the entire glycerol utilization (gylABX) operon of Streptomyces coelicolor A3(2) and identification of a closely associated transcription unit

The entire glycerol utilization (gylABX) operon of Streptomyces coelicolor A3(2) was cloned and its transcriptional organization and regulation was analyzed by Northern blotting, S1 nuclease mapping and transcriptional fusions. Transcription of the operon is glycerol-inducible and glucose-repressible; glyA (presumptively encoding glycerol kinase), gylB (encoding sn-glycerol-3-phosphate dehydrogenase) and gylX (a non-essential 1.1 kb sequence) are transcribed consecutively to give a 5.4 kb mRNA. Two alternative transcription termination or gyl mRNA processing sites are located within the operon; one (a discrete site) lies between gylB and gylX and the other (a heterogeneous site) positioned 3 kb into the operon, may correspond to the gylA-gylB intercistronic region. A 0.9 kb glycerol-inducible transcription unit is located immediately upstream of gylABX. Transcriptional fusion studies employing an attP site-deleted phage vector provided complementary evidence for the organization of the operon.

Evolution of alpha q- and beta-tubulin genes as inferred by the nucleotide sequences of sea urchin cDNA clones.

Evolutionary studies on the tubulin multigene families were initiated by nucleotide sequence analysis of cDNA clones complementary to sea urchin (Lytechinus pictus) testis alpha- and beta-tubulin cDNA clones (p beta 1, p beta 2, p beta e) demonstrated the existence of tubulin mRNA heterogeneity. p beta 2 and p beta 3 contain identical tubulin-coding regions and extremely similar 3' untranslated sequences, including a polyadenylation signal (AAUAAA). However, p beta 2 contains an additional region of 3' untranslated sequence which includes a second polyadenylation signal. These two sequences may be allelic, representing products of alternative transcription termination or processing pathways. p beta 1 and p beta 2 (or p beta 3) cDNAs almost certainly correspond to transcripts of distinct but evolutionarily related genes. Examination of the available coding portions showed that they differ only by a few silent nucleotide substitutions and the deletion/insertion of one codon; most of the differences are clustered within the last 15 3'-end codons. In contrast, their 3' untranslated sequences are considerably divergent. Nucleotide alignment in this region was feasible by considering specific point and segmental mutations, mainly T in equilibrium or formed from C transitions and small deletions/insertions associated with small direct repeats. The sea urchin alpha- and beta-tubulin cDNA and corresponding protein sequences were compared with previously described tubulin cDNA and protein sequences from other organisms. Both alpha and beta tubulins are very conserved proteins, evolving with a rate comparable to that of histones. Analysis of the nucleotide divergence of the coding cDNA regions showed that replacement sites have changed with a rate 20-175 times lower than that of the silent sites. Among the 177 codons compared between the sea urchin testis and chick brain beta-tubulin cDNAs, there are 7 conservative amino acid replacements and the deletion/insertion of two codons. Most of these changes are clustered near the C-terminus. The 161-amino acid portion of chick brain, rat and porcine alpha-tubulin sequences differs by 3 conservative amino acid replacements from the corresponding sea urchin testis alpha-tubulin sequence. The compared interspecies 3' untranslated sequences are very divergent.