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RNA Biology Dec 2021RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of...
RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins via proximity-based biotinylation. RPL applied to identified proteins involved in both canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5'-3' proximity and unexplored subcellular localizations of poly(A) RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA-protein interactions important for health and disease.
Topics: Ascorbate Peroxidases; Biotinylation; CRISPR-Associated Proteins; CRISPR-Cas Systems; HEK293 Cells; Humans; Poly A; RNA; RNA, Guide, CRISPR-Cas Systems; RNA, Small Nuclear; RNA-Binding Proteins; Recombinant Fusion Proteins; Staining and Labeling
PubMed: 34006179
DOI: 10.1080/15476286.2021.1917215 -
Plant & Cell Physiology Mar 2020Regulation of the stability and the quality of mitochondrial RNA is essential for the maintenance of mitochondrial and cellular functions in eukaryotes. We have...
Regulation of the stability and the quality of mitochondrial RNA is essential for the maintenance of mitochondrial and cellular functions in eukaryotes. We have previously reported that the eukaryotic poly(A)-specific ribonuclease (PARN) and the prokaryotic poly(A) polymerase encoded by AHG2 and AGS1, respectively, coordinately regulate the poly(A) status and the stability of mitochondrial mRNA in Arabidopsis. Mitochondrial function of PARN has not been reported in any other eukaryotes. To know how much this PARN-based mitochondrial mRNA regulation is conserved among plants, we studied the AHG2 and AGS1 counterparts of the liverwort, Marchantia polymorpha, a member of basal land plant lineage. We found that M. polymorpha has one ortholog each for AHG2 and AGS1, named MpAHG2 and MpAGS1, respectively. Their Citrine-fused proteins were detected in mitochondria of the liverwort. Molecular genetic analysis showed that MpAHG2 is essential and functionally interacts with MpAGS1 as observed in Arabidopsis. A recombinant MpAHG2 protein had a deadenylase activity in vitro. Overexpression of MpAGS1 and the reduced expression of MpAHG2 caused an accumulation of polyadenylated Mpcox1 mRNA. Furthermore, MpAHG2 suppressed Arabidopsis ahg2-1 mutant phenotype. These results suggest that the PARN-based mitochondrial mRNA regulatory system is conserved in land plants.
Topics: Arabidopsis; Embryophyta; Exoribonucleases; Gene Expression Regulation, Plant; Marchantia; Mitochondria; Phenotype; Plants, Genetically Modified; Poly A; RNA, Messenger; RNA, Mitochondrial; Transcriptome
PubMed: 31722408
DOI: 10.1093/pcp/pcz212 -
Scientific Reports Nov 2018Nocturnin (NOCT) helps the circadian clock to adjust metabolism according to day and night activity. NOCT is upregulated in early evening and it has been proposed that...
Nocturnin (NOCT) helps the circadian clock to adjust metabolism according to day and night activity. NOCT is upregulated in early evening and it has been proposed that NOCT serves as a deadenylase for metabolic enzyme mRNAs. We present a 2.7-Å crystal structure of the catalytic domain of human NOCT. Our structure shows that NOCT has a close overall similarity to CCR4 deadenylase family members, PDE12 and CNOT6L, and to a DNA repair enzyme TDP2. All the key catalytic residues present in PDE12, CNOT6L and TDP2 are conserved in NOCT and have the same conformations. However, we observe substantial differences in the surface properties of NOCT, an unexpectedly narrow active site pocket, and conserved structural elements in the vicinity of the catalytic center, which are unique to NOCT and absent in the deadenylases PDE12/CNOT6L. Moreover, we show that in contrast to human PDE12 and CNOT6L, NOCT is completely inactive against poly-A RNA. Our work thus reveals the structure of an intriguing circadian protein and suggests that NOCT has considerable differences from the related deadenylases, which may point to a unique cellular function of this enzyme.
Topics: Catalytic Domain; DNA-Binding Proteins; Exoribonucleases; Nuclear Proteins; Phosphoric Diester Hydrolases; Poly A; RNA, Messenger; Recombinant Proteins; Ribonucleases; Transcription Factors
PubMed: 30389976
DOI: 10.1038/s41598-018-34615-0 -
Frontiers in Bioengineering and... 2022The generation of the stable, high-level recombinant protein-producing cell lines remains a significant challenge in the biopharmaceutical industry. Expression vector...
The generation of the stable, high-level recombinant protein-producing cell lines remains a significant challenge in the biopharmaceutical industry. Expression vector optimization is an effective strategy to increase transgene expression levels and stability, and the choice of suitable poly A element is crucial for the expression of recombinant protein. In this study, we investigated the effects of different poly A elements on transgene expression in Chinese hamster ovary (CHO) cells. Five poly A elements, including bovine growth hormone (BGH), mutant BGH, herpes simplex virus type 1 thymidine kinase (HSV-TK), SV40, and a synthetic (Synt) poly A, were cloned into the expression vector and transfected into CHO cells. The results indicated the SV40 and Synt poly A sequences can significant improve transgene expression in stable transfected CHO cells and maintain long-term expression. However, qPCR results showed that the expression at protein level was not related to the gene copy number and mRNA level. Importantly, the SV40 and Synt poly A elements decreased the variation of transgene expression. Furthermore, it also showed that the SV40 and Synt poly A elements induced higher levels of adalimumab expression. In conclusion, SV40 poly A and Synt poly A are stronger elements that increase stable transgene expression and decrease the variation of expression, and the choice of suitable poly A element is helpful to improve the expression of recombinant protein.
PubMed: 35141210
DOI: 10.3389/fbioe.2022.722722 -
Science (New York, N.Y.) Feb 2021Polyadenylate [poly(A)] tail addition to the 3' end of a wide range of RNAs is a highly conserved modification that plays a central role in cellular RNA function....
Polyadenylate [poly(A)] tail addition to the 3' end of a wide range of RNAs is a highly conserved modification that plays a central role in cellular RNA function. Elements for nuclear expression (ENEs) are cis-acting RNA elements that stabilize poly(A) tails by sequestering them in RNA triplex structures. A crystal structure of a double ENE from a rice hAT transposon messenger RNA complexed with poly(A) at a resolution of 2.89 angstroms reveals multiple modes of interaction with poly(A), including major-groove triple helices, extended minor-groove interactions with RNA double helices, a quintuple-base motif that transitions poly(A) from minor-groove associations to major-groove triple helices, and a poly(A) 3'-end binding pocket. Our findings both expand the repertoire of motifs involved in long-range RNA interactions and provide insights into how polyadenylation can protect an RNA's extreme 3' end.
Topics: Crystallization; Nucleic Acid Conformation; Oryza; Poly A; Polyadenylation; RNA Stability; RNA, Messenger
PubMed: 33414189
DOI: 10.1126/science.abe6523 -
ELife Aug 2020Yeast cells undergoing the diauxic response show a striking upstream shift in poly(A) site utilization, with increased use of ORF-proximal poly(A) sites resulting in...
Yeast cells undergoing the diauxic response show a striking upstream shift in poly(A) site utilization, with increased use of ORF-proximal poly(A) sites resulting in shorter 3' mRNA isoforms for most genes. This altered poly(A) pattern is extremely similar to that observed in cells containing Pol II derivatives with slow elongation rates. Conversely, cells containing derivatives with fast elongation rates show a subtle downstream shift in poly(A) sites. Polyadenylation patterns of many genes are sensitive to both fast and slow elongation rates, and a global shift of poly(A) utilization is strongly linked to increased purine content of sequences flanking poly(A) sites. Pol II processivity is impaired in diauxic cells, but strains with reduced processivity and normal Pol II elongation rates have normal polyadenylation profiles. Thus, Pol II elongation speed is important for poly(A) site selection and for regulating poly(A) patterns in response to environmental conditions.
Topics: Poly A; Polyadenylation; RNA Polymerase II; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Elongation, Genetic
PubMed: 32845240
DOI: 10.7554/eLife.59810 -
Molecular and Cellular Biology Sep 2022The 3' ends of eukaryotic mRNAs are generated by cleavage of nascent transcripts followed by polyadenylation, which occurs at numerous sites within 3' untranslated...
The 3' ends of eukaryotic mRNAs are generated by cleavage of nascent transcripts followed by polyadenylation, which occurs at numerous sites within 3' untranslated regions (3' UTRs) but rarely within coding regions. An individual gene can yield many 3'-mRNA isoforms with distinct half-lives. We dissect the relative contributions of protein-coding sequences (open reading frames [ORFs]) and 3' UTRs to polyadenylation profiles in yeast. ORF-deleted derivatives often display strongly decreased mRNA levels, indicating that ORFs contribute to overall mRNA stability. Poly(A) profiles, and hence relative isoform half-lives, of most (9 of 10) ORF-deleted derivatives are very similar to their wild-type counterparts. Similarly, in-frame insertion of a large protein-coding fragment between the ORF and 3' UTR has minimal effect on the poly(A) profile in all 15 cases tested. Last, reciprocal ORF/3'-UTR chimeric genes indicate that the poly(A) profile is determined by the 3' UTR. Thus, 3' UTRs are self-contained modular entities sufficient to determine poly(A) profiles and relative 3'-isoform half-lives. In the one atypical instance, ORF deletion causes an upstream shift of poly(A) sites, likely because juxtaposition of an unusually high AT-rich stretch directs polyadenylation closely downstream. This suggests that long AT-rich stretches, which are not encountered until after coding regions, are important for restricting polyadenylation to 3' UTRs.
Topics: 3' Untranslated Regions; 5' Untranslated Regions; Poly A; Polyadenylation; Protein Isoforms; RNA Isoforms; RNA, Messenger; Saccharomyces cerevisiae
PubMed: 35972270
DOI: 10.1128/mcb.00244-22 -
IScience Sep 2023Cell differentiation is associated with global changes in translational activity. Here, we characterize how mRNA poly(A) tail processing supports this dynamic. We...
Cell differentiation is associated with global changes in translational activity. Here, we characterize how mRNA poly(A) tail processing supports this dynamic. We observe that decreased translation during neuronal differentiation of P19 cells correlates with the downregulation of 5'-terminal oligopyrimidine (TOP) transcripts which encode the translational machinery. Despite their downregulation, TOP transcripts remain highly stable and show increased translation as cells differentiate. Changes in TOP mRNA metabolism are reflected by their accumulation with poly(A) tails ∼60-nucleotide (nt) long. The dynamic changes in poly(A) processing can be partially recapitulated by depleting LARP1 or activating the mTOR pathway in undifferentiated cells. Although mTOR-induced accumulation of TOP mRNAs with tails ∼60-nt long does not trigger differentiation, it is associated with reduced proliferation of neuronal progenitors. We propose that while TOP mRNAs are transcriptionally silenced, their post-transcriptional regulation mediated by a specific poly(A) processing ensures an adequate supply of ribosomes to complete differentiation.
PubMed: 37636056
DOI: 10.1016/j.isci.2023.107511 -
BMC Genomics Sep 2023While numerous studies have described the transcriptomes of extracellular vesicles (EVs) in different cellular contexts, these efforts have typically relied on...
BACKGROUND
While numerous studies have described the transcriptomes of extracellular vesicles (EVs) in different cellular contexts, these efforts have typically relied on sequencing methods requiring RNA fragmentation, which limits interpretations on the integrity and isoform diversity of EV-targeted RNA populations. It has been assumed that mRNA signatures in EVs are likely to be fragmentation products of the cellular mRNA material, and the extent to which full-length mRNAs are present within EVs remains to be clarified.
RESULTS
Using long-read nanopore RNA sequencing, we sought to characterize the full-length polyadenylated (poly-A) transcriptome of EVs released by human chronic myelogenous leukemia K562 cells. We detected 443 and 280 RNAs that were respectively enriched or depleted in EVs. EV-enriched poly-A transcripts consist of a variety of biotypes, including mRNAs, long non-coding RNAs, and pseudogenes. Our analysis revealed that 10.58% of all EV reads, and 18.67% of all cellular (WC) reads, corresponded to known full-length transcripts, with mRNAs representing the largest biotype for each group (EV = 58.13%, WC = 43.93%). We also observed that for many well-represented coding and non-coding genes, diverse full-length transcript isoforms were present in EV specimens, and these isoforms were reflective-of but often in different ratio compared to cellular samples.
CONCLUSION
This work provides novel insights into the compositional diversity of poly-A transcript isoforms enriched within EVs, while also underscoring the potential usefulness of nanopore sequencing to interrogate secreted RNA transcriptomes.
Topics: Humans; Nanopore Sequencing; Transcriptome; Extracellular Vesicles; RNA; RNA, Messenger; Poly A
PubMed: 37736705
DOI: 10.1186/s12864-023-09552-6 -
Genome Biology Sep 2019Polyadenylation plays a key role in producing mature mRNAs in eukaryotes. It is widely believed that the poly(A)-binding proteins (PABs) uniformly bind to poly(A)-tailed...
BACKGROUND
Polyadenylation plays a key role in producing mature mRNAs in eukaryotes. It is widely believed that the poly(A)-binding proteins (PABs) uniformly bind to poly(A)-tailed mRNAs, regulating their stability and translational efficiency.
RESULTS
We observe that the homozygous triple mutant of broadly expressed Arabidopsis thaliana PABs, AtPAB2, AtPAB4, and AtPAB8, is embryonic lethal. To understand the molecular basis, we characterize the RNA-binding landscape of these PABs. The AtPAB-binding efficiency varies over one order of magnitude among genes. To identify the sequences accounting for the variation, we perform poly(A)-seq that directly sequences the full-length poly(A) tails. More than 10% of poly(A) tails contain at least one guanosine (G); among them, the G-content varies from 0.8 to 28%. These guanosines frequently divide poly(A) tails into interspersed A-tracts and therefore cause the variation in the AtPAB-binding efficiency among genes. Ribo-seq and genome-wide RNA stability assays show that AtPAB-binding efficiency of a gene is positively correlated with translational efficiency rather than mRNA stability. Consistently, genes with stronger AtPAB binding exhibit a greater reduction in translational efficiency when AtPAB is depleted.
CONCLUSIONS
Our study provides a new mechanism that translational efficiency of a gene can be regulated through the G-content-dependent PAB binding, paving the way for a better understanding of poly(A) tail-associated regulation of gene expression.
Topics: Arabidopsis; Arabidopsis Proteins; Base Composition; Gene Expression Regulation, Plant; Genes, Plant; Guanosine; Poly(A)-Binding Protein II; Poly(A)-Binding Proteins; Protein Binding; Protein Biosynthesis; RNA, Messenger
PubMed: 31481099
DOI: 10.1186/s13059-019-1799-8