How Gene Endings are Predefined by Promoters: The Start is also the Finish

Every gene in our DNA has a beginning and an end. Correct identification of the gene terminators is critical in the production of a functional protein. Much research has been done to determine what determines when, where, and at what site on the DNA a gene “starts”. But where the end of a gene is is is a different story – selection of transcription termination sites is presumed to depend on downstream elements and external factors.

In their most recent study published in the journal cell, researchers from the Max Planck Institute for Immunology and Epigenetics made the surprising discovery that for most of our genes, the site of the start of transcription determines the site of the end of transcription. This phenomenon is well conserved across species and predetermines mRNA end sites at the start of transcription, and plays an important role in cell identity and function.

All cells in an organism contain an identical DNA sequence. What determines the identity and function of individual cells and tissues is the set of genes that will be active in a particular place, at a particular time. These active genes are transcribed from the DNA template into distinct messenger RNA (mRNA) molecules and will encode proteins that the cell needs to function.

At specific sites called promoters, a complex molecular machinery begins transcribing DNA sequences into mRNA. Interestingly, most genes contain several potential sites where transcription can start or end. This means that for each gene, depending on the start or end site, the mRNAs can be different. Expression of a single gene in different variants expands the diversity and functionality of the genome many times over. At the same time, it adds another layer of complexity to the study of the genome.

RNA snapshots from start to finish

Scientists at the Max Planck Institute for Immunology and Epigenetics in Freiburg wanted to know how many different start and end sites each gene uses, in which combination, and whether the combinations are different in different circumstances. says Valerie Hilgers, a research group leader at MPI-IE.

The scientists used a modified next-generation sequencing technology to read the individual mRNAs. For conventional short read sequencing, each mRNA is broken down into shorter fragments that are amplified and then sequenced to produce the read. Bioinformatics techniques are then used to piece the readings together like a jigsaw, in a continuous sequence.

To obtain full-length mRNA information for the entire genome in several Drosophila tissues, including the brain, Hilgers collaborated with the MPI Deep Sequencing Facility to improve specific long-read sequencing techniques. “Long-read sequencing allows the retrieval of much longer sequence reads than standard widely used sequencing. However, we had to improve this technique and increase the sample read length several-fold to obtain full-length mRNA information in our various model systems,” says Carlos Alfonso Gonzalez, first author of the publication. .

In addition to fruit flies, Hilgers’ lab also included a human model of the nervous system in their study: cerebral organoids—”mini-brains” cultured in a dish of iPS cells. Transcriptional end sites were previously identified at the beginning of transcription.

Aggregated data representing all mRNA at the whole-molecule scale gives unprecedented insight into the transcription of individual genes. Transcription start sites are specifically associated with distinct transcription end sites,” says Hilgers.

This association is in fact causal: in the ovaries, for example, the artificial activation of TSS that is normally used only in the brain bypasses the natural TES and artificially induces the use of brain TES. This illustrates the critical role of TSS in shaping the unique RNA landscape of each tissue, and thus influencing tissue identity.

Promoter dominance leads to RNA diversity, gene function, and tissue identity

However, one phenomenon stood out. “Some TSSs show unpredictable dominant behavior. They abolish conventional signals for transcription termination, overexpress other TSSs, and cause selection of distinct TESs. Accordingly, we named them dominant promoters,” says Alfonso Gonzalez.

Furthermore, the team found that the interactions between these dominant stimuli and their associated gene endings were directed by distinct gene signatures. Importantly, findings in Drosophila brain cells can be replicated in human brain organelles, demonstrating that promoter dominance is a conserved, and possibly universal, mechanism for regulating the production of functional proteins and cell functions.

What could be the physiological significance of this new mechanism? Through an in-depth analysis of sequence conservation, the Freiburg researchers discovered that TSSs and TESs show co-evolution: Over millions of years of evolution between species, changes of single nucleotides in a gene at dominant promoters were initiated by changes at the corresponding end of the gene. .

“We interpret this observation as a push through evolution, to maintain interaction between the two ends of the gene, which indicates the great importance of these links for animal fitness,” says Valerie Hilgers.