scholarly journals Long-read sequencing of nascent RNA reveals coupling among RNA processing events

2018 ◽  
Vol 28 (7) ◽  
pp. 1008-1019 ◽  
Author(s):  
Lydia Herzel ◽  
Korinna Straube ◽  
Karla M. Neugebauer
Keyword(s):  
Rna Processing ◽  
Long Read ◽  
Nascent Rna

Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus

1991 ◽  
Vol 11 (6) ◽  
pp. 3180-3190
Author(s):  
J Huang ◽  
L H van der Ploeg
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Processing ◽  
Rnase A ◽  
Chain Elongation ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Transcriptional Elongation ◽  
Splice Acceptor ◽  
Trans Splicing ◽  
Nascent Rna

Numerous protein-coding genes of the protozoan Trypanosoma brucei are arranged in tandem arrays that are transcribed polycistronically. The pre-mRNA transcripts are processed by trans splicing, leading to the addition of a capped 39-nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at the hsp70 locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at the hsp70 locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3' end of hsp70 nascent RNA with a short (about 180-nt) 32P tail. The structure of the labeled nascent hsp70 RNA could then be analyzed by RNase T1 and RNase T1/RNase A mapping. We show that the trans splicing of hsp70 pre-mRNA did not occur immediately after the synthesis of the 3' splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3' splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5' end that mapped to the polyadenylation site of the hsp70 genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascent hsp70 RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly(A) addition.

scholarly journals Revealing nascent RNA processing dynamics with nano-COP

2021 ◽  
Author(s):  
Heather L. Drexler ◽  
Karine Choquet ◽  
Hope E. Merens ◽  
Paul S. Tang ◽  
Jared T. Simpson ◽  
...  
Keyword(s):  
Rna Processing ◽  
Nascent Rna

scholarly journals Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus.

1991 ◽  
Vol 11 (6) ◽  
pp. 3180-3190 ◽  
Author(s):  
J Huang ◽  
L H van der Ploeg
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Processing ◽  
Rnase A ◽  
Chain Elongation ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Transcriptional Elongation ◽  
Splice Acceptor ◽  
Trans Splicing ◽  
Nascent Rna

Numerous protein-coding genes of the protozoan Trypanosoma brucei are arranged in tandem arrays that are transcribed polycistronically. The pre-mRNA transcripts are processed by trans splicing, leading to the addition of a capped 39-nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at the hsp70 locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at the hsp70 locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3' end of hsp70 nascent RNA with a short (about 180-nt) 32P tail. The structure of the labeled nascent hsp70 RNA could then be analyzed by RNase T1 and RNase T1/RNase A mapping. We show that the trans splicing of hsp70 pre-mRNA did not occur immediately after the synthesis of the 3' splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3' splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5' end that mapped to the polyadenylation site of the hsp70 genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascent hsp70 RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly(A) addition.

scholarly journals Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide

2016 ◽  
Vol 11 (3) ◽  
pp. 413-428 ◽  
Author(s):  
Takayuki Nojima ◽  
Tomás Gomes ◽  
Maria Carmo-Fonseca ◽  
Nicholas J Proudfoot
Keyword(s):  
Rna Processing ◽  
Genome Wide ◽  
Nascent Rna

scholarly journals Preparation of Mammalian Nascent RNA for Long Read Sequencing

2020 ◽  
Vol 133 (1) ◽  
Author(s):  
Kirsten A. Reimer ◽  
Karla M. Neugebauer
Keyword(s):  
Long Read ◽  
Nascent Rna

scholarly journals POINT Technology Illuminates the Processing of Polymerase-Associated Intact Nascent Transcripts

2020 ◽  
Author(s):  
Rui Sousa-Luis ◽  
Gwendal Dujardin ◽  
Inna Zukher ◽  
Hiroshi Kimura ◽  
Maria Carmo-Fonseca ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Rna Degradation ◽  
List Type ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Human Genes ◽  
Nascent Rna ◽  
Full Length Transcript ◽  
Nascent Transcripts

SUMMARYMammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5’ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5’P-RNA at polyadenylation sites. Furthermore POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.HIGHLIGHTSPOINT methodology dissects intact nascent RNA processingSpecificity of Xrn2 exonuclease in co-transcriptional RNA degradationSplicing suppresses Xrn2-dependent premature terminationDifferent kinetic classes of co-transcriptional splicing in human genes

scholarly journals Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing

2019 ◽  
Author(s):  
Alexander M. Price ◽  
Katharina E. Hayer ◽  
Alexa B.R. McIntyre ◽  
Nandan S. Gokhale ◽  
Jonathan S. Abebe ◽  
...  
Keyword(s):  
Rna Processing ◽  
Viral Pathogen ◽  
Short Read Sequencing ◽  
Long Read ◽  
Splicing Efficiency ◽  
A New Technique ◽  
Nucleotide Resolution ◽  
And Function ◽  
Infectious Cycle

AbstractAdenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, we profiled m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.

scholarly journals Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander M. Price ◽  
Katharina E. Hayer ◽  
Alexa B. R. McIntyre ◽  
Nandan S. Gokhale ◽  
Jonathan S. Abebe ◽  
...  
Keyword(s):  
Rna Processing ◽  
Viral Pathogen ◽  
Short Read Sequencing ◽  
Long Read ◽  
Splicing Efficiency ◽  
A New Technique ◽  
Nucleotide Resolution ◽  
And Function ◽  
Infectious Cycle

AbstractAdenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, here we profile m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.

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