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Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation.

Roy CK, Olson S, Graveley BR, Zamore PD, Moore MJ - Elife (2015)

Bottom Line: Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten.To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules.Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

View Article: PubMed Central - PubMed

Affiliation: RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States.

ABSTRACT
Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA-DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

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Related in: MedlinePlus

SeqZip assay to measure endogenous mRNA isoform expression.(A) The SeqZip strategy to detect human CD45 mRNA isoforms. (B) Denaturing PAGE gels showing products of reverse transcriptase (RT) (top left) or SeqZip (bottom left) CD45 mRNA obtained from two different human Jurkat and U-937 T-cell lines, or a 1:1 mixture of the two. Top right: quantified band intensities from gels at left. Bottom right: mirrored lane profiles from the mix lanes (RT—left; SeqZip—right). (C) The six possible combinations of EDA (blue; + or −) and V (light blue; 120, 95 and 0) alternative splicing within mouse Fn1 transcripts. Filled boxes depict exons, diagonal lines indicate isoform sequences not shown, and straight lines show absence of exon(s) in the final mRNA. (D) Detailed schematic of ligamer pools used to analyze indicated regions of Fn1 RNA. (E) SeqZip ligation products from immortalized MEFs with indicated Fn1 genotypes. Radioactive PCR separated on a native acrylamide gel. (F) Fn1 isoform abundance measured by SeqZip and PacBio. Black bars indicate observed individual exon (‘Individual Pool’; EDA, V) or combination frequencies (‘Combination A + V pool’, [EDA, V]). Shown in light gray are expected combination isoform intensities, and where available, the frequency of PacBio reads (mid-gray, lower bars).DOI:http://dx.doi.org/10.7554/eLife.03700.008
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fig3: SeqZip assay to measure endogenous mRNA isoform expression.(A) The SeqZip strategy to detect human CD45 mRNA isoforms. (B) Denaturing PAGE gels showing products of reverse transcriptase (RT) (top left) or SeqZip (bottom left) CD45 mRNA obtained from two different human Jurkat and U-937 T-cell lines, or a 1:1 mixture of the two. Top right: quantified band intensities from gels at left. Bottom right: mirrored lane profiles from the mix lanes (RT—left; SeqZip—right). (C) The six possible combinations of EDA (blue; + or −) and V (light blue; 120, 95 and 0) alternative splicing within mouse Fn1 transcripts. Filled boxes depict exons, diagonal lines indicate isoform sequences not shown, and straight lines show absence of exon(s) in the final mRNA. (D) Detailed schematic of ligamer pools used to analyze indicated regions of Fn1 RNA. (E) SeqZip ligation products from immortalized MEFs with indicated Fn1 genotypes. Radioactive PCR separated on a native acrylamide gel. (F) Fn1 isoform abundance measured by SeqZip and PacBio. Black bars indicate observed individual exon (‘Individual Pool’; EDA, V) or combination frequencies (‘Combination A + V pool’, [EDA, V]). Shown in light gray are expected combination isoform intensities, and where available, the frequency of PacBio reads (mid-gray, lower bars).DOI:http://dx.doi.org/10.7554/eLife.03700.008

Mentions: Shown are various uses of SeqZip toward multi-site sequence investigation of RNA. ‘Product Length Adjustment’ has applications similar to those shown in Figure 3E, where isoform discrimination solely on the basis of size separation of RT-PCR products would be ambiguous; with SeqZip, the lengths of individual products can be adjusted through ligamer design. ‘RNA barcoding’ depicts the introduction of randomized rather than static barcodes, allowing for molecular indexing or amplification bias estimation. ‘Quantify RNA-integrity’ relies on the requirement of molecular continuity between sites of ligamer hybridization in order to obtain a SeqZip product (check mark). If the intervening sequences are not intact, no product is obtained (X). Thus, SeqZip can be used to monitor the integrity of long RNAs. ‘Multi-site SNP detection’ is described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The panel depicting ‘Introduction of destruction sequences’ illustrates how short DNA oligos targeting ligamer-specific barcodes between hybridization regions (in this case ‘B’) could be useful in the selective cleavage and destruction of particular ligation products. In the example shown, the ABC ligamer product would be cleaved with a restriction enzyme targeting the double-stranded oligo:barcode, while DEF would be left intact for downstream applications. ‘Sequence discovery using combined SeqZip and Reverse Transcription’ illustrates 5′ end sequence discovery using Cap Analysis of Gene Expression combined with SeqZip ligamers. This allows one to investigate novel 5′ end sequence connections to distant 3′ sequences. ‘Multi-site AS QPCR analysis’ is also described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The essential benefit over a conventional QPCR workflow is that SeqZip compresses distant sequences into a QPCR-friendly amplicon size and reduces the number of required primers.


Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation.

Roy CK, Olson S, Graveley BR, Zamore PD, Moore MJ - Elife (2015)

SeqZip assay to measure endogenous mRNA isoform expression.(A) The SeqZip strategy to detect human CD45 mRNA isoforms. (B) Denaturing PAGE gels showing products of reverse transcriptase (RT) (top left) or SeqZip (bottom left) CD45 mRNA obtained from two different human Jurkat and U-937 T-cell lines, or a 1:1 mixture of the two. Top right: quantified band intensities from gels at left. Bottom right: mirrored lane profiles from the mix lanes (RT—left; SeqZip—right). (C) The six possible combinations of EDA (blue; + or −) and V (light blue; 120, 95 and 0) alternative splicing within mouse Fn1 transcripts. Filled boxes depict exons, diagonal lines indicate isoform sequences not shown, and straight lines show absence of exon(s) in the final mRNA. (D) Detailed schematic of ligamer pools used to analyze indicated regions of Fn1 RNA. (E) SeqZip ligation products from immortalized MEFs with indicated Fn1 genotypes. Radioactive PCR separated on a native acrylamide gel. (F) Fn1 isoform abundance measured by SeqZip and PacBio. Black bars indicate observed individual exon (‘Individual Pool’; EDA, V) or combination frequencies (‘Combination A + V pool’, [EDA, V]). Shown in light gray are expected combination isoform intensities, and where available, the frequency of PacBio reads (mid-gray, lower bars).DOI:http://dx.doi.org/10.7554/eLife.03700.008
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4442144&req=5

fig3: SeqZip assay to measure endogenous mRNA isoform expression.(A) The SeqZip strategy to detect human CD45 mRNA isoforms. (B) Denaturing PAGE gels showing products of reverse transcriptase (RT) (top left) or SeqZip (bottom left) CD45 mRNA obtained from two different human Jurkat and U-937 T-cell lines, or a 1:1 mixture of the two. Top right: quantified band intensities from gels at left. Bottom right: mirrored lane profiles from the mix lanes (RT—left; SeqZip—right). (C) The six possible combinations of EDA (blue; + or −) and V (light blue; 120, 95 and 0) alternative splicing within mouse Fn1 transcripts. Filled boxes depict exons, diagonal lines indicate isoform sequences not shown, and straight lines show absence of exon(s) in the final mRNA. (D) Detailed schematic of ligamer pools used to analyze indicated regions of Fn1 RNA. (E) SeqZip ligation products from immortalized MEFs with indicated Fn1 genotypes. Radioactive PCR separated on a native acrylamide gel. (F) Fn1 isoform abundance measured by SeqZip and PacBio. Black bars indicate observed individual exon (‘Individual Pool’; EDA, V) or combination frequencies (‘Combination A + V pool’, [EDA, V]). Shown in light gray are expected combination isoform intensities, and where available, the frequency of PacBio reads (mid-gray, lower bars).DOI:http://dx.doi.org/10.7554/eLife.03700.008
Mentions: Shown are various uses of SeqZip toward multi-site sequence investigation of RNA. ‘Product Length Adjustment’ has applications similar to those shown in Figure 3E, where isoform discrimination solely on the basis of size separation of RT-PCR products would be ambiguous; with SeqZip, the lengths of individual products can be adjusted through ligamer design. ‘RNA barcoding’ depicts the introduction of randomized rather than static barcodes, allowing for molecular indexing or amplification bias estimation. ‘Quantify RNA-integrity’ relies on the requirement of molecular continuity between sites of ligamer hybridization in order to obtain a SeqZip product (check mark). If the intervening sequences are not intact, no product is obtained (X). Thus, SeqZip can be used to monitor the integrity of long RNAs. ‘Multi-site SNP detection’ is described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The panel depicting ‘Introduction of destruction sequences’ illustrates how short DNA oligos targeting ligamer-specific barcodes between hybridization regions (in this case ‘B’) could be useful in the selective cleavage and destruction of particular ligation products. In the example shown, the ABC ligamer product would be cleaved with a restriction enzyme targeting the double-stranded oligo:barcode, while DEF would be left intact for downstream applications. ‘Sequence discovery using combined SeqZip and Reverse Transcription’ illustrates 5′ end sequence discovery using Cap Analysis of Gene Expression combined with SeqZip ligamers. This allows one to investigate novel 5′ end sequence connections to distant 3′ sequences. ‘Multi-site AS QPCR analysis’ is also described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The essential benefit over a conventional QPCR workflow is that SeqZip compresses distant sequences into a QPCR-friendly amplicon size and reduces the number of required primers.

Bottom Line: Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten.To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules.Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

View Article: PubMed Central - PubMed

Affiliation: RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States.

ABSTRACT
Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA-DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

Show MeSH
Related in: MedlinePlus