Thoughts on NGS, alternative splicing and what we still need to know.

Twenty-two years ago I attended my first scientific meeting: the 1993 Cold Spring Harbor RNAmeeting. Although initially overwhelmed by not being able to keep up with the seemingly endless knowledge of others, I left the meeting both having fallen in love with the field of RNA and having met many people who continue to be some of my closest friends and colleagues. However, what I didn’t realize at the time was that the most important thing to happen at that meeting was not the 2nd-year grad student poster I presented, or even the shaping of my career path, but rather the formalizing of the formation of the RNA Society. For though I was just learning these lessons, many others already knew that RNA is sufficiently exciting and full of unexplored mysteries to warrant a lifetime of study, and that research is both more enjoyable andmore efficient when done in the context of a community. The founders of the Society also recognized the importance of having a journal to encourage and promote the publication of progress made on the study of RNA, and so this journal aptly named RNA was born two years later. Excitingly, the assumption made 22 years ago that RNA would continue to interest and amaze us could not have been more accurate. These past two decades have witnessed an explosion in our knowledge of almost all aspects of RNA, including the achievement of high resolution structures of the ribosome, the recognition that alternative splicing is the rule not the exception, and the discovery that our cells are full of countless non-coding RNAs that play roles in almost every aspect of gene regulation and cellular function. Whereas the study of RNA used to be confined to a relative few RNA-centric research groups, now seemingly everyone is interested and there is hardly a laboratory around in which someone isn’t carrying out some study related in some way to RNA. As one of my senior colleagues once quipped “we [outside the RNA field] used to think of RNA like we think of water—present and essential, but not really that interesting. Now we know better.” Much of the recent explosion in our knowledge of RNA can be traced to the development of genomic profiling methods, starting with microarrays and then taking off exponentially with the advent of next generation sequencing (NGS). The NGS methods, in particular, have allowed for the identification of RNA populations we never guessed existed: miRNAs, lncRNAs, lincRNAs, piRNAs, circRNA, to name but a few. The discoveries of these classes of RNA have opened exciting new areas of biology and have captured the attention of the science community broadly. Closer to my own area of research, NGS methods have also revealed there to be much greater variation and regulation in RNA processing events than previously imagined. For example, when I started in the field of splicing regulation, leading reviews predicted that alternative splicing might occur in as much as 5% of human genes. Current estimates are now that all but 5% of human genes undergo some form of alternative splicing. While all of this new insight has been tremendously exciting, the speed of discovery has left many gaps and holes in its wake. As we move into the post-NGS era I see two major understudied areas, at least with regards to alternative splicing, that represent the greatest challenge and the greatest opportunity for impact in the decade ahead: understanding the functional consequence of alternative splicing and determining the molecular mechanism(s) by which the spliceosome activity is directed to preferentially form one splice isoform over another. The discovery that virtually all mammalian genes are alternatively spliced in some cell type or condition is of course extremely exciting for those of us interested in splicing regulation—and is a compelling statistic to cite in publications or grants to underscore the importance of studying this process. Yet the fact remains the functional relevance of alternative splicing has only been demonstrated for an exceedingly small fraction of these cases. To truly appreciate the full impact of alternative splicing on biologic processes, and argue against those who wonder if it might all be “noise,” we need to do better. The question is how to achieve this goal. It is clearly