Sites of transcription initiation drive mRNA isoform selection

Alfonso-Gonzalez, Carlos; Legnini, Ivano; Holec, Sarah; Arrigoni, Laura; Ozbulut, Hasan Can; Mateos, Fernando; Koppstein, David; Rybak-Wolf, Agnieszka; Bönisch, Ulrike; Rajewsky, Nikolaus; Hilgers, Valérie

doi:10.1016/j.cell.2023.04.012

Cited by 35 publications

(13 citation statements)

References 113 publications

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“…Such isoforms are often low abundance transcripts, are found in many genomes and may well determine the site of 3′ end formation (Alfonso-Gonzalez et al, 2023;Nguyen et al, 2014;Thomas et al, 2020;Wang et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

“…One such low abundance unannotated transcript, initiating upstream of HMS2 and extending over 2 kb through the 3′UTR, is characterised here to validate the potential of sfNET‐Seq to discover new transcription units. Such isoforms are often low abundance transcripts, are found in many genomes and may well determine the site of 3′ end formation (Alfonso‐Gonzalez et al, 2023; Nguyen et al, 2014; Thomas et al, 2020; Wang et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

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Size fractionated NET‐Seq reveals a conserved architecture of transcription units around yeast genes

Xi,

Nguyen,

Murray

et al. 2024

Yeast

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Genomes from yeast to humans are subject to pervasive transcription. A single round of pervasive transcription is sufficient to alter local chromatin conformation, nucleosome dynamics and gene expression, but is hard to distinguish from background signals. Size fractionated native elongating transcript sequencing (sfNET‐Seq) was developed to precisely map nascent transcripts independent of expression levels. RNAPII‐associated nascent transcripts are fractionation into different size ranges before library construction. When anchored to the transcription start sites (TSS) of annotated genes, the combined pattern of the output metagenes gives the expected reference pattern. Bioinformatic pattern matching to the reference pattern identified 9542 transcription units in Saccharomyces cerevisiae, of which 47% are coding and 53% are noncoding. In total, 3113 (33%) are unannotated noncoding transcription units. Anchoring all transcription units to the TSS or polyadenylation site (PAS) of annotated genes reveals distinctive architectures of linked pairs of divergent transcripts approximately 200nt apart. The Reb1 transcription factor is enriched 30nt downstream of the PAS only when an upstream (TSS −60nt with respect to PAS) noncoding transcription unit co‐occurs with a downstream (TSS +150nt) coding transcription unit and acts to limit levels of upstream antisense transcripts. The potential for extensive transcriptional interference is evident from low abundance unannotated transcription units with variable TSS (median −240nt) initiating within a 500nt window upstream of, and transcribing over, the promoters of protein‐coding genes. This study confirms a highly interleaved yeast genome with different types of transcription units altering the chromatin landscape in distinctive ways, with the potential to exert extensive regulatory control.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Size fractionated NET‐Seq reveals a conserved architecture of transcription units around yeast genes

Xi,

Nguyen,

Murray

et al. 2024

Yeast

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“…A second terminator with additional and/or differing binding motifs for members of the polyadenylation complex could therefore interact more strongly with transcription factors bound to the 5′ end, resulting in more efficient gene looping and gene expression (Andreou et al 2021 ). In Drosophila, a connection was found between the transcriptional start site (TSS) of a gene and the choice of poly(A)site, resulting in preferred TSS–poly(A) site pairs, which differed depending on the analysed tissue (Alfonso-Gonzalez et al 2023 ). However, in Physcomitrella, we observed the effect of double terminators on gene expression but found no indication for synergistic promoter–terminator effects.…”

Section: Discussionmentioning

confidence: 99%

Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella

Niederau,

Eglé,

Willig

et al. 2024

Plant Cell Rep

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Key message Characterization of Physcomitrella 3′UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming. Abstract The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular, the good manufacturing practice (GMP)-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features, such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3′UTRs (untranslated regions) of which 7964 transcripts contain at least one intron. Over 91% of 3′UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3′UTRs, 14 terminator candidates were selected and characterized via transient Dual-Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3′UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than tenfold, demonstrating the stronger effect promoters play as compared to terminators. Among selected terminator attributes, the number of polyadenylation sites as well as polyadenylation signals were found to influence terminator performance the most. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.

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“…A second terminator with additional and/or differing binding motifs for members of the polyadenylation complex could therefore interact more strongly with transcription factors bound to the 5' end, resulting in more efficient gene looping and gene expression (Andreou et al, 2021). In Drosophila, a connection was found between the transcriptional start site (TSS) of a gene and the choice of poly(A)site, resulting in preferred TSS -poly(A) site pairs, which differed depending on the analysed tissue (Alfonso-Gonzalez et al, 2023).…”

Section: Rhoadesmentioning

confidence: 99%

Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella

Niederau,

Eglé,

Willig

et al. 2023

Preprint

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The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular the GMP-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized new terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3 prime UTRs (untranslated regions) of which 7,964 transcripts contain at least one intron. Over 91% of 3 prime UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3 prime UTRs, 14 terminator candidates were selected and characterized via transient Dual Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3 prime UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1,000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than 10-fold, demonstrating the stronger effect promoters play as compared to terminators. The number of polyadenylation sites as well as polyadenylation signals were found to be major determinants of terminator performance. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.

show abstract

Sites of transcription initiation drive mRNA isoform selection

Cited by 35 publications

References 113 publications

Size fractionated NET‐Seq reveals a conserved architecture of transcription units around yeast genes

Size fractionated NET‐Seq reveals a conserved architecture of transcription units around yeast genes

Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella

Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella

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