scholarly journals Antibody cross-reactivity accounts for widespread appearance of m1A in 5’ UTRs

2019 ◽  
Author(s):  
Anya V. Grozhik ◽  
Anthony O. Olarerin-George ◽  
Miriam Sindelar ◽  
Xing Li ◽  
Steven S. Gross ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Binding Sites ◽  
Cross Reactivity ◽  
Rna Seq ◽  
Transcription Start ◽  
Single Nucleotide ◽  
Biochemical Assays ◽  
Binding Antibody ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractN1-methyladenosine (m1A) was recently identified as a new mRNA modification based on its mapping to the 5’ UTRs of thousands of mRNAs with an m1A-binding antibody. More recent studies have confirmed the prevalence of m1A, while others have questioned it. To address this discrepancy, we mapped m1A using ultra-deep RNA-Seq datasets based on m1A-induced misincorporations during reverse transcription. Using this approach, we find m1A only in the mitochondrial MT-ND5 transcript. In contrast, when we mapped m1A antibody-binding sites at single-nucleotide resolution, we found binding to transcription start nucleotides in mRNA 5’ UTRs. Using different biochemical assays, we find that m1A is not present at these sites. Instead, we find that the m1A antibody exhibits m1A-independent binding to mRNA cap structures. We also tested a new and independently derived m1A antibody. We show that this m1A antibody lacks m7G cap-binding cross-reactivity, and notably does not map to 5’ UTRs in the transcriptome. Our data demonstrate that high-stoichiometry m1A sites are rare in the transcriptome and that previous mapping of m1A to mRNA 5’ UTRs are due to unintended binding of the m1A antibody to m7G cap structure in mRNA.

scholarly journals Antibody cross-reactivity accounts for widespread appearance of m1A in 5’UTRs

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Anya V. Grozhik ◽  
Anthony O. Olarerin-George ◽  
Miriam Sindelar ◽  
Xing Li ◽  
Steven S. Gross ◽  
...  
Keyword(s):  
Cross Reactivity ◽  
Start Site ◽  
Rna Seq ◽  
Transcription Start ◽  
Single Nucleotide ◽  
Alternative Approach ◽  
Binding Antibody ◽  
Nucleotide Resolution ◽  
Bioinformatic Approach ◽  
Single Nucleotide Resolution

Abstract N1-methyladenosine (m1A) was proposed to be a highly prevalent modification in mRNA 5’UTRs based on mapping studies using an m1A-binding antibody. We developed a bioinformatic approach to discover m1A and other modifications in mRNA throughout the transcriptome by analyzing preexisting ultra-deep RNA-Seq data for modification-induced misincorporations. Using this approach, we detected appreciable levels of m1A only in one mRNA: the mitochondrial MT-ND5 transcript. As an alternative approach, we also developed an antibody-based m1A-mapping approach to detect m1A at single-nucleotide resolution, and confirmed that the commonly used m1A antibody maps sites to the transcription-start site in mRNA 5’UTRs. However, further analysis revealed that these were false-positives caused by binding of the antibody to the m7G-cap. A different m1A antibody that lacks cap-binding cross-reactivity does not show enriched binding in 5’UTRs. These results demonstrate that high-stoichiometry m1A sites are exceedingly rare in mRNAs and that previous mappings of m1A to 5’UTRs were the result of antibody cross-reactivity to the 5’ cap.

scholarly journals Function annotation of the rice transcriptome at single-nucleotide resolution by RNA-seq

2010 ◽  
Vol 20 (9) ◽  
pp. 1238-1249 ◽  
Author(s):  
T. Lu ◽  
G. Lu ◽  
D. Fan ◽  
C. Zhu ◽  
W. Li ◽  
...  
Keyword(s):  
Rna Seq ◽  
Single Nucleotide ◽  
Function Annotation ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals Comparative analysis of LIN28-RNA binding sites identified at single nucleotide resolution

RNA Biology ◽  
2017 ◽  
Vol 14 (12) ◽  
pp. 1756-1765 ◽  
Author(s):  
Elizabeth Ransey ◽  
Anders Björkbom ◽  
Victor S. Lelyveld ◽  
Przemyslaw Biecek ◽  
Lorena Pantano ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Binding Sites ◽  
Rna Binding ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution ◽  
Rna Binding Sites

scholarly journals Single nucleotide resolution RNA-seq uncovers new regulatory mechanisms in the opportunistic pathogen Streptococcus agalactiae

BMC Genomics ◽  
2015 ◽  
Vol 16 (1) ◽  
Author(s):  
Isabelle Rosinski-Chupin ◽  
Elisabeth Sauvage ◽  
Odile Sismeiro ◽  
Adrien Villain ◽  
Violette Da Cunha ◽  
...  
Keyword(s):  
Streptococcus Agalactiae ◽  
Opportunistic Pathogen ◽  
Regulatory Mechanisms ◽  
Rna Seq ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals TIF-Seq2 disentangles overlapping isoforms in complex human transcriptomes

2020 ◽  
Vol 48 (18) ◽  
pp. e104-e104 ◽  
Author(s):  
Jingwen Wang ◽  
Bingnan Li ◽  
Sueli Marques ◽  
Lars M Steinmetz ◽  
Wu Wei ◽  
...  
Keyword(s):  
Human Cell Line ◽  
Rna Seq ◽  
Single Nucleotide ◽  
Rna Molecules ◽  
Unannotated Transcript ◽  
Long Read ◽  
Nucleotide Resolution ◽  
Overlapping Transcripts ◽  
Single Nucleotide Resolution ◽  
Leukaemia Patient

Abstract Eukaryotic transcriptomes are complex, involving thousands of overlapping transcripts. The interleaved nature of the transcriptomes limits our ability to identify regulatory regions, and in some cases can lead to misinterpretation of gene expression. To improve the understanding of the overlapping transcriptomes, we have developed an optimized method, TIF-Seq2, able to sequence simultaneously the 5′ and 3′ ends of individual RNA molecules at single-nucleotide resolution. We investigated the transcriptome of a well characterized human cell line (K562) and identified thousands of unannotated transcript isoforms. By focusing on transcripts which are challenging to be investigated with RNA-Seq, we accurately defined boundaries of lowly expressed unannotated and read-through transcripts putatively encoding fusion genes. We validated our results by targeted long-read sequencing and standard RNA-Seq for chronic myeloid leukaemia patient samples. Taking the advantage of TIF-Seq2, we explored transcription regulation among overlapping units and investigated their crosstalk. We show that most overlapping upstream transcripts use poly(A) sites within the first 2 kb of the downstream transcription units. Our work shows that, by paring the 5′ and 3′ end of each RNA, TIF-Seq2 can improve the annotation of complex genomes, facilitate accurate assignment of promoters to genes and easily identify transcriptionally fused genes.

scholarly journals Profiling the binding sites of RNA-binding protein by LACE-seq

2021 ◽  
Author(s):  
Ruibao Su ◽  
Di Wang ◽  
Changchang Cao ◽  
Yuanchao Xue
Keyword(s):  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Transcription Termination ◽  
Early Embryo ◽  
Linear Amplification ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Abstract RNA-binding proteins (RBPs) directly interact with various RNAs in living cells to regulate their processing, translation, and stability. Identifying the precise binding sites of RBPs is critical for appreciating their physiological or pathological roles in germline and early embryo development. Current methods typically need millions of cells to map RBP binding positions, which prevents us from appreciating the crucial role of RBPs in early development. Here, we present the LACE-seq method for unbiased mapping of RBP-binding sites at single-nucleotide resolution in fewer cells or even single oocytes. LACE-seq depends on RBP-mediated reverse transcription termination, and linear amplification of the cDNA ends for deep sequencing. To further promote its application, we describe a step-by-step protocol about how to construct a successful LACE-seq library.

Sign in / Sign up

Export Citation Format

Share Document