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Assessment of Highly Complex Alternative Splicing of Neurexins Performed with SMRT Sequencing

Wednesday, March 19, 2014

A new paper in the Proceedings of the National Academy of Sciences from the laboratories of Stephen R. Quake and Thomas C. Südhof (both at Stanford University) describes the direct, full-length transcript sequencing of RNA molecules that are essential to synapse formation in the mammalian brain. The team used Single Molecule, Real-Time (SMRT®) Sequencing to analyze full-length mRNAs from different members of the neurexin gene family and used that information to examine alternative splicing events.

In the publication entitled “Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing,” the scientists highlight the importance of understanding alternative splicing in neurexins. “Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available,” they write. The alternative splice isoforms are differentially regulated in different brain regions, exhibit a diurnal cycle, and are modulated by development, neurotrophins, and neuronal activity.

Prior to SMRT Sequencing, the authors write that “despite extensive studies, the full extent of neurexin alternative splicing remains unclear.” Neurexin isoforms have been previously analyzed with full-length cDNA sequencing and PCR analysis, but “only a small fraction of these isoforms were actually identified in sequenced full-length cDNAs,” the authors note. “The relatively large size of α-neurexin transcripts (~4–5 kb) has made it difficult to obtain information about their full-length sequence, and hence about the use of alternative splice sites within single transcripts.”

For this study, researchers used the PacBio® platform to sequence transcripts generated by three neurexin genes in adult mice. “Read lengths of up to 30 kb enabled us to identify all of the splice combinations within a single transcript,” they report. With sequencing reads representing more than 25,000 full-length mRNAs, the team made several important discoveries. These include: a novel alternatively spliced exon; even higher isoform diversity than was anticipated; and the finding that splicing events seem to occur independently of one another. The team was able to map out the full transcript landscape for a neurexin gene, showing alternative splicing at all six canonical sites as well as at several noncanonical sites.

Being able to directly assess alternative splicing not only provided evidence for suspected isoform diversity, but also revealed “that neurexins are likely even more polymorphic than previously thought,” the team reports. Based on their observations from SMRT Sequencing, they calculated how many neurexin variants were possible in total. “We observed in this manner a minimal diversity of 1,159 isoforms for Nrxn1α, 1,120 isoforms for Nrxn3α, and a total of 152 isoforms for all three β-neurexins,” they write. “Thus, earlier estimates of 2,000–3,000 neurexin variants created by alternative splicing may have been an underestimate, because our present study arrived at the same numbers by analyzing only one brain region and one developmental stage.”

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