Nuclear mRNA Quality Control and Cytoplasmic NMD Are Linked by the Guard Proteins Gbp2 and Hrb1.

Pre-mRNA splicing is critical for cells, as defects in this process can lead to altered open reading frames and defective proteins, potentially causing neurodegenerative diseases and cancer. Introns are removed in the nucleus and splicing is documented by the addition of exon-junction-complexes (EJCs) at exon-exon boundaries. This "memory" of splicing events is important for the ribosome, which translates the RNAs in the cytoplasm. In case a stop codon was detected before an EJC, translation is blocked and the RNA is eliminated by the nonsense-mediated decay (NMD). In the model organism Saccharomyces cerevisiae, two guard proteins, Gbp2 and Hrb1, have been identified as nuclear quality control factors for splicing. In their absence, intron-containing mRNAs leak into the cytoplasm. Their presence retains transcripts until the process is completed and they release the mRNAs by recruitment of the export factor Mex67. On transcripts that experience splicing problems, these guard proteins recruit the nuclear RNA degradation machinery. Interestingly, they continue their quality control function on exported transcripts. They support NMD by inhibiting translation and recruiting the cytoplasmic degradation factors. In this way, they link the nuclear and cytoplasmic quality control systems. These discoveries are also intriguing for humans, as homologues of these guard proteins are present also in multicellular organisms. Here, we provide an overview of the quality control mechanisms of pre-mRNA splicing, and present Gbp2 and Hrb1, as well as their human counterparts, as important players in these pathways.

Nuclear SR-protein mediated mRNA quality control is continued in cytoplasmic nonsense-mediated decay.

One important task of eukaryotic cells is to translate only mRNAs that were correctly processed to prevent the production of truncated proteins, found in neurodegenerative diseases and cancer. Nuclear quality control of splicing requires the SR-like proteins Gbp2 and Hrb1 in S. cerevisiae, where they promote the degradation of faulty pre-mRNAs. Here we show that Gbp2 and Hrb1 also function in nonsense mediated decay (NMD) of spliced premature termination codon (PTC)-containing mRNAs. Our data support a model in which they are in a complex with the Upf-proteins and help to transmit the Upf1-mediated PTC recognition to the transcripts ends. Most importantly they appear to promote translation repression of spliced transcripts that contain a PTC and to finally facilitate degradation of the RNA, presumably by supporting the recruitment of the degradation factors. Therefore, they seem to control mRNA quality beyond the nuclear border and may thus be global surveillance factors. Identification of SR-proteins as general cellular surveillance factors in yeast will help to understand the complex human system in which many diseases with defects in SR-proteins or NMD are known, but the proteins were not yet recognized as general RNA surveillance factors.

Evolution of intron splicing towards optimized gene expression is based on various Cis- and Trans-molecular mechanisms.

Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when organisms adapt to new challenges? How does evolution optimize inefficiency of introns' splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron's splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein's domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands.