黑料吃瓜资源

Cold Spring Harbor, NY 鈥� No one knows how many times in a day, or even an hour, the trillions of cells in our body need to make proteins. But we do know that it鈥檚 going on all the time, on a massive scale. We also know that every time this happens, an editing process takes place in the cell nucleus. Called RNA splicing, it makes sure that the RNA 鈥渋nstructions鈥� sent to cellular protein factories correspond precisely with the blueprint encoded in our genes.

Researchers led by Adrian Krainer, a 黑料吃瓜资源 (CSHL) Professor, and Assistant Professor Justin Kinney, are teasing out the rules that guide how cells process these RNA messages, enabling better predictions about the impact of specific genetic mutations that affect this process. This in turn will help assess how certain mutations affect a person鈥檚 risk for disease.

Splicing removes interrupting segments called introns from the raw, unedited RNA copy of a gene, leaving only the exons, or protein-coding regions. There are over 200,000 introns in the human genome, and if they are spliced out imprecisely, cells will generate faulty proteins. The results can be life-threatening: about 14% of the single-letter mutations that have been linked to human diseases are thought to occur within the DNA sequences that flag intron positions in the genome.

The cell鈥檚 splicing machinery seeks 鈥渟plice sites鈥� to correctly remove introns from a raw RNA message. Splice sites throughout the genome are similar but not identical, and small changes don鈥檛 always impair splicing efficiency. For the splice site at the beginning of an intron鈥攌nown as its 5鈥� [鈥渇ive-prime鈥漖 splice site, Krainer says, 鈥渨e know that at the first and second [DNA-letter] position, mutations have a very strong impact. Mutations elsewhere in the intron can have dramatic effects or no effect, or something in between.鈥�

That鈥檚 made it hard to predict how mutations at splice sites within disease-linked genes will impact patients. For example, mutations in the genes BRCA1 or BRCA2 can increase a woman鈥檚 risk of breast and ovarian cancer, but not every mutation is harmful.

In experiments led by first author Mandy Wong, a Krainer lab postdoc, the team created 5鈥� splice sites with every possible combination of DNA letters, then measured how well the associated introns were removed from a larger piece of RNA. For their experiments, they used introns from three disease-associated genes鈥擝RCA2 and two genes in which mutations cause neurodegenerative diseases, IKBKAP and SMN1.

In one intron of each of the three genes, the team tested over 32,000 5鈥� splice sites. They found that specific DNA sequences corresponded with similar splicing efficiency or inefficiency in different introns. This is a step toward making general predictions. But they also found that other features of each gene鈥攖he larger context鈥攖ended to modify the impact in each specific case. In other words: how a mutation within a given 5鈥� splice site will affect splicing is somewhat predictable, but is also influenced by context beyond the splice site itself.

Krainer says this knowledge will better help predict the impact of splice-site mutations鈥攂ut a deeper investigation is needed.

Written by: Jennifer Michalowski, Science Writer | [email protected] | 516-367-8455

Funding

NIH-NIGMS; NIH CSHL Cancer Center Support Grant.

Citation

Wong MS et al, 鈥淨uantitative activity profile and context dependence of all human 5鈥� splice sites,鈥� is available online ahead of print and will appear in the September 2018 issue of Molecular Cell.