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Massively parallel reporter assay of 3’UTR sequences identifies in vivo rules for mRNA degradation

ABSTRACT: The stability of mRNAs is regulated by signals within their sequences, but a systematic and predictive understanding of the underlying sequence rules remains elusive. Here, we introduce UTR-Seq, a combination of massively parallel reporter assays and regression models, to survey the dynamics of tens-of-thousands of 3’UTR sequences during early zebrafish embryogenesis. UTR-Seq revealed two temporal degradation programs: a maternally encoded early-onset program and a late-onset program that accelerated degradation after zygotic genome activation. Three signals regulated early-onset rates: stabilizing poly-U and UUAG sequences, and destabilizing GC-rich signals. Three signals explained late-onset degradation: miR-430 seeds, AU-rich sequences and Pumilio recognition sites. Sequence based regression models translated 3’UTRs into their unique decay patterns, and predicted the in vivo impact of sequence signals on mRNA stability. Their application led to the successful design of artificial 3’UTRs that conferred specific mRNA dynamics. UTR-Seq provides a general strategy to uncover the rules of RNA cis-regulation.

ORGANISM(S): Danio rerio

PROVIDER: GSE106677 | GEO | 2017/12/12

SECONDARY ACCESSION(S): PRJNA417597

REPOSITORIES: GEO

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Massively parallel reporter assay of 3’UTR sequences identifies in vivo rules for mRNA degradation

Project description:Massively parallel reporter assay of 3’UTR sequences identifies in vivo rules for mRNA degradation

| PRJNA417597 | ENA

Gene Expression differences between replication error deficient and proficient colorectal cancers: the dominant role of deletions in 3âUTR poly T sequences

Project description:16 replication error proficient (RER-/MSI-) and 14 replication error deficient (RER+/MSI+) colorectal cancer cell lines Replication error deficient (RER+) colorectal cancers are a distinct subset of colorectal cancers, characterised by inactivation of the DNA mismatch repair system. They are typically pseudodiploid, accumulate mutations in repetitive sequences as a result of their mismatch repair deficiency, and have distinct pathologies. Regulatory sequences controlling all aspects of mRNA processing, including especially message stability are found in the 3âUTR sequence of most genes. The relevant sequences are typically A/U rich elements and/or U repeats. Microarray analysis of 14 RER+ (deficient) and 16 RER- (proficient) colorectal cancer (CRC) cell lines confirms a striking difference in expression profiles. Analysis of the incidence of mononucleotide repeat sequences in the 3âUTRs, 5âUTRs and coding sequences of those genes most differentially expressed in RER+ versus â cell lines has shown that much of this differential expression can be explained by the occurrence of a massive enrichment of genes with 3âUTR T repeats longer than 11 base pairs in the most differentially expressed genes. This enrichment was confirmed by analysis of two published consensus sets of RER differentially expressed probe sets for a large number of primary colorectal cancers. Sequence analysis of the 3âUTRs of a selection of the most differentially expressed genes shows that they all contain deletions in these repeats in all RER+ cell lines studied. These data strongly imply that deregulation of mRNA stability through accumulation of mutations in repetitive regulatory 3âUTR sequences underlies the striking difference in expression profiles between RER+ and RER- colorectal cancers. 16 replication error proficient (RER-/MSI-) and 14 replication error deficient (RER+/MSI+) colorectal cancer cell lines.

2010-10-19 | E-GEOD-24795 | biostudies-arrayexpress

Gene Expression differences between replication error deficient and proficient colorectal cancers: the dominant role of deletions in 3’UTR poly T sequences

Project description:16 replication error proficient (RER-/MSI-) and 14 replication error deficient (RER+/MSI+) colorectal cancer cell lines Replication error deficient (RER+) colorectal cancers are a distinct subset of colorectal cancers, characterised by inactivation of the DNA mismatch repair system. They are typically pseudodiploid, accumulate mutations in repetitive sequences as a result of their mismatch repair deficiency, and have distinct pathologies. Regulatory sequences controlling all aspects of mRNA processing, including especially message stability are found in the 3’UTR sequence of most genes. The relevant sequences are typically A/U rich elements and/or U repeats. Microarray analysis of 14 RER+ (deficient) and 16 RER- (proficient) colorectal cancer (CRC) cell lines confirms a striking difference in expression profiles. Analysis of the incidence of mononucleotide repeat sequences in the 3’UTRs, 5’UTRs and coding sequences of those genes most differentially expressed in RER+ versus – cell lines has shown that much of this differential expression can be explained by the occurrence of a massive enrichment of genes with 3’UTR T repeats longer than 11 base pairs in the most differentially expressed genes. This enrichment was confirmed by analysis of two published consensus sets of RER differentially expressed probe sets for a large number of primary colorectal cancers. Sequence analysis of the 3’UTRs of a selection of the most differentially expressed genes shows that they all contain deletions in these repeats in all RER+ cell lines studied. These data strongly imply that deregulation of mRNA stability through accumulation of mutations in repetitive regulatory 3’UTR sequences underlies the striking difference in expression profiles between RER+ and RER- colorectal cancers.

2010-10-19 | GSE24795 | GEO

Human 5′ UTR design and variant effect prediction from a massively parallel translation assay

Project description:Predicting the impact of cis-regulatory sequence on gene expression is a foundational challenge for biology. We combine polysome profiling of hundreds of thousands of randomized 5′ UTRs with deep learning to build a predictive model that relates human 5′ UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5 UTRs that accurately target specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,000 truncated human 5′ UTRs and 3,577 naturally-occurring variants and show that the model accurately predicts ribosome loading of these sequences. Finally, we provide evidence of 47 SNVs associated with human diseases that cause a significant change in ribosome loading and thus a plausible molecular basis for disease.

2018-05-03 | GSE114002 | GEO

Learning the regulatory grammar of yeast 5' untranslated regions from a large library of random sequences

Project description:Our ability to predict protein expression from DNA sequence alone remains poor, reflecting our limited understanding of cis-regulatory grammar and hampering the design of engineered genes for synthetic biology applications. Here, we generate a model that predicts the translational efficiency of the 5’ untranslated region (UTR) of mRNAs in the yeast Saccharomyces cerevisiae. We constructed a library of half a million 50-nucleotide- long random 5’ UTRs and assayed their activity in a massively parallel growth selection experiment. The resulting data allow us to quantify the impact on translation of Kozak sequence composition, upstream open reading frames (uORFs) and secondary structure. We trained a convolutional neural network (CNN) on the random library and showed that it performs well at predicting the translational efficiency of both a held-out set of the random 5’ UTRs as well as native S. cerevisiae 5’ UTRs. The model additionally was used to computationally evolve highly translating 5’ UTRs. We confirmed experimentally that the great majority of the evolved sequences lead to higher translation rates than the starting sequences, demonstrating the predictive power of this model. The sequences were assayed in the context of a constant promoter and downstream coding sequence. Cell growth was used as a proxy for protein expression. Growth was measured by determining the ratio of sequence counts before and after growth selection.

2017-09-27 | GSE104252 | GEO

Optimizing 5’UTRs for mRNA-delivered gene editing using deep learning

Project description:mRNA therapeutics are revolutionizing the pharmaceutical industry, but methods to optimize the primary sequence for increased expression are still lacking. Here, we design 5’UTRs for efficient mRNA translation using deep learning. We perform polysome profiling of fully or partially randomized 5'UTR libraries in three cell types and find that UTR performance is highly correlated across cell types. We train models on all our datasets and use them to guide the design of high-performing 5’UTRs using gradient descent and generative neural networks. We experimentally test designed 5’UTRs with mRNA encoding megaTALTM gene editing enzymes for two different gene targets and in two different cell lines. We find that the designed 5’UTRs support strong gene editing activity. Editing efficiency is correlated between cell types and gene targets, although the best performing UTR was specific to one cargo and cell type. Our results highlight the potential of model-based sequence design for mRNA therapeutics.

2023-07-01 | GSE232927 | GEO

Cytoplasmic cleavage of IMPA1 3’UTR is necessary for maintaining axon integrity.

Project description:The 3’ untranslated regions (3’UTRs) of messenger RNAs (mRNA) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localisation and translation. Incorrect processing and delivery of mRNA causes severe developmental defects and has been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3’UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3’UTR, and stable 3’UTR-derived fragments of unknown function. Cleavage of the long 3’UTR of Inositol Monophosphatase 1 ( IMPA1 ), mediated by a protein complex containing the endonuclease Ago2 and the RNA binding protein HuD, generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a new mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates a yet undescribed class of non-coding RNAs.

2020-12-31 | GSE160025 | GEO

High-throughput discovery of post-transcriptional cis-regulatory elements (miRNA-seq)

Project description:Post-transcriptional gene regulation controls the amount of a protein produced from an individual mRNA transcript by altering mRNA decay and translation rates. Many putative post-transcriptional cis-regulatory elements have been identified from computational, molecular biology, and biochemical studies studies; however, identifying which sequence elements are sufficient to regulate expression remains challenging. We created a high-throughput, cell-based screen that tested the post-transcriptional regulatory potential for thousands of short sequence elements. Sequences with known effects have the expected performance in this screen, showing this methodology is robust. Hundreds of novel short sequences were identified as being able to alter gene expression, both by increasing and decreasing protein production from the fluorescence reporter, and we validated the effects for fifty of these sequences. Importantly, sequences discovered in this screen are conserved in human 3′UTRs, and furthermore, the sequences can regulate expression in the context of those endogenous 3′UTRs. Hundreds of previously unknown post-transcriptional cis-regulatory elements exist, many of which increase gene expression. These results suggest that each human 3′UTR has many small cis-regulatory elements that interact with RNA binding proteins, and these interactions control the fate of an mRNA transcript.

2016-03-07 | GSE75136 | GEO

High-throughput discovery of post-transcriptional cis-regulatory elements (8-nt)

Project description:Post-transcriptional gene regulation controls the amount of a protein produced from an individual mRNA transcript by altering mRNA decay and translation rates. Many putative post-transcriptional cis-regulatory elements have been identified from computational, molecular biology, and biochemical studies studies; however, identifying which sequence elements are sufficient to regulate expression remains challenging. We created a high-throughput, cell-based screen that tested the post-transcriptional regulatory potential for thousands of short sequence elements. Sequences with known effects have the expected performance in this screen, showing this methodology is robust. Hundreds of novel short sequences were identified as being able to alter gene expression, both by increasing and decreasing protein production from the fluorescence reporter, and we validated the effects for fifty of these sequences. Importantly, sequences discovered in this screen are conserved in human 3?UTRs, and furthermore, the sequences can regulate expression in the context of those endogenous 3?UTRs. Hundreds of previously unknown post-transcriptional cis-regulatory elements exist, many of which increase gene expression. These results suggest that each human 3?UTR has many small cis-regulatory elements that interact with RNA binding proteins, and these interactions control the fate of an mRNA transcript.

2016-03-07 | GSE75135 | GEO

Cellular stress alters 3’UTR landscape through alternative polyadenylation and isoform-specific degradation

Project description:Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different lengths of 3’ untranslated region (3’UTR). Here we show arsenic stress elicits global shortening of 3’UTRs through two mechanisms. First, stress leads to immediate shortening of 3’UTR due to preferential usage of proximal cleavage and polyadenylation sites (PASs), as revealed by 3’ end sequencing of newly made RNAs that are metabolically labeled with 4-thiouridine. Second, long 3’UTR isoforms are more rapidly degraded during recovery from stress as compared to short 3’UTR isoforms, further shortening 3’UTR lengths in the cell. Using ribonucleoprotein immunoprecipitation coupled with 3’ end sequencing (3’READS+RIP), we show that the RNA-binding protein T cell-restricted intracellular antigen-1 (Tia1) preferentially interacts with long 3’UTR isoforms via U-rich elements in alternative 3’UTR sequences, and the interaction correlates with stress granule (SG) association during stress and with mRNA decay during recovery from stress, indicating SG-mediated RNA clearance mechanism post stress. Importantly, genes whose 3’UTRs are shortened by APA during stress can evade stress-induced 3’UTR size-based mRNA degradation, leading to higher transcript abundance post stress. Moreover, proliferating and differentiated cells display different extents of 3’UTR shortening after stress, indicating cell type-specific of impact of stress on the 3’UTR landscape. Together, our data indicate that 3’UTR length plays important roles in gene expression in stressed cells, and APA functions as an adaptive stress response mechanism to preserve mRNAs.

2018-06-13 | GSE101851 | GEO

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