EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates

Welch, Joshua D.; Slevin, Michael K.; Tatomer, Deirdre C.; Duronio, Robert J.; Prins, Jan F.; Marzluff, William F.

doi:10.1261/rna.048785.114

Cited by 22 publications

(48 citation statements)

References 41 publications

(69 reference statements)

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“…The very 3 ′ end of the consensus binding site determined by CLIP-seq differs from the genomic sequence, with an enrichment of uridine in the last 2 nt. Recently, it was reported that a fraction of all histone mRNAs have the last 2-3 ′ encoded nucleotides replaced with uridines posttranscriptionally (Welch et al 2015) and we detected these 3 ′ ends present in the cell in our HITS-CLIP experiments.…”

Section: Analysis Of Crosslinking Datasupporting

confidence: 62%

“…Note that the x axis is the same for the boxplots (E,F) and the seqlogos (G,H) to display the sequence composition of the nuclease-resistant histone SL RNA fragments. (I) We used the AppEnD tool (Welch et al 2015) to identify nontemplated tails on the 3 ′ ends of histone RNA molecules present in HITS-CLIP reads. To avoid calling sequencing errors as tails, only reads that extended at least 4 nt into the 3 ′ adapter were analyzed.…”

Section: Mapping Inferred Nuclease Cleavage Sites Precisely Defines Tmentioning

confidence: 99%

“…Addition of uridines at the 3 ′ end of histone mRNA is the first step in 3 ′ -5 ′ degradation of histone transcripts (Slevin et al 2014). Moreover, many histone mRNAs that are not undergoing degradation during S phase have lost 1-2 nt from their 3 ′ end, which have been replaced by one or two uridines to restore the length of the histone mRNA (Welch et al 2015). These are precisely the positions at which the U's are overrepresented in the HITS-CLIP RNA fragment consensus sequence (positions 39 and 40 in Fig.…”

Section: Mapping Inferred Nuclease Cleavage Sites Precisely Defines Tmentioning

confidence: 99%

“…4G,H). We used the AppEnD tool (Welch et al 2015) to define the 3 ′ end of each transcript and directly search for nontemplated 3 ′ additions in the SLBP HITS-CLIP reads. This analysis revealed many nontemplated additions ( Fig.…”

Section: Mapping Inferred Nuclease Cleavage Sites Precisely Defines Tmentioning

confidence: 99%

“…We used the AppEnD tool (Welch et al 2015) to identify nontemplated 3 ′ additions in SLBP HITS-CLIP reads. Briefly, AppEnD identifies nontemplated 3 ′ additions by aligning a read to a reference genome, detecting the position within the read where the read sequence stops matching the genome sequence, and removing the portion of this mismatched sequence that comes from a sequencing adapter (if any).…”

Section: Identifying Nontemplated 3 ′ Additions In Hits-clip Readsmentioning

confidence: 99%

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A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs

Brooks

Lyons

Mahoney

et al. 2015

RNA

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The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stemloop (SL) structure. This SL structure interacts with the stem-loop binding protein (SLBP), which is involved in all aspects of RDhistone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3 ′ hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3 ′ end of RD-histone mRNAs together with SLBP.

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Section: Analysis Of Crosslinking Datasupporting

confidence: 62%

Section: Mapping Inferred Nuclease Cleavage Sites Precisely Defines Tmentioning

confidence: 99%

Section: Mapping Inferred Nuclease Cleavage Sites Precisely Defines Tmentioning

confidence: 99%

Section: Mapping Inferred Nuclease Cleavage Sites Precisely Defines Tmentioning

confidence: 99%

Section: Identifying Nontemplated 3 ′ Additions In Hits-clip Readsmentioning

confidence: 99%

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A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs

Brooks

Lyons

Mahoney

et al. 2015

RNA

Self Cite

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RNA uridylation: a key posttranscriptional modification shaping the coding and noncoding transcriptome

et al. 2017

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RNA uridylation is a potent and widespread posttranscriptional regulator of gene expression. RNA uridylation has been detected in a range of eukaryotes including trypanosomes, animals, plants, and fungi, but with the noticeable exception of budding yeast. Virtually all classes of eukaryotic RNAs can be uridylated and uridylation can also tag viral RNAs. The untemplated addition of a few uridines at the 3 0 end of a transcript can have a decisive impact on RNA's fate. In rare instances, uridylation is an intrinsic step in the maturation of noncoding RNAs like for the U6 spliceosomal RNA or mitochondrial guide RNAs in trypanosomes. Uridylation can also switch specific miRNA precursors from a degradative to a processing mode. This switch depends on the number of uridines added which is regulated by the cellular context. Yet, the typical consequence of uridylation on mature noncoding RNAs or their precursors is to accelerate decay. Importantly, mRNAs are also tagged by uridylation. In fact, the advent of novel high throughput sequencing protocols has recently revealed the pervasiveness of mRNA uridylation, from plants to humans. As for noncoding RNAs, the main function to date for mRNA uridylation is to promote degradation. Yet, additional roles begin to be ascribed to U-tailing such as the control of mRNA deadenylation, translation control and possibly storage. All these new findings illustrate that we are just beginning to appreciate the diversity of roles played by RNA uridylation and its full temporal and spatial implication in regulating gene expression.

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Measuring the tail: Methods for poly(A) tail profiling

et al. 2022

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The 3 0 -end poly(A) tail is an important and potent feature of most mRNA molecules that affects mRNA fate and translation efficiency. Polyadenylation is a posttranscriptional process that occurs in the nucleus by canonical poly(A) polymerases (PAPs). In some specific instances, the poly(A) tail can also be extended in the cytoplasm by noncanonical poly(A) polymerases (ncPAPs). This epitranscriptomic regulation of mRNA recently became one of the most interesting aspects in the field. Advances in RNA sequencing technologies and software development have allowed the precise measurement of poly(A) tails, identification of new ncPAPs, expansion of the function of known enzymes, discovery and a better understanding of the physiological role of tail heterogeneity, and recognition of a correlation between tail length and RNA translatability. Here, we summarize the development of polyadenylation research methods, including classic low-throughput approaches, Illumina-based genomewide analysis, and advanced state-of-art techniques that utilize long-read third-generation sequencing with Pacific Biosciences and Oxford Nanopore Technologies platforms. A boost in technical opportunities over recent decades has allowed a better understanding of the regulation of gene expression at the mRNA level. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico K E Y W O R D S 3 0 -end tailing, poly(A) tail, regulation of gene expression, RNA, RNA sequencing

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EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates

Cited by 22 publications

References 41 publications

A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs

A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs

RNA uridylation: a key posttranscriptional modification shaping the coding and noncoding transcriptome

Measuring the tail: Methods for poly(A) tail profiling

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