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- Assessing the involvement of Dis3L1 in mammalian quality control pathwaysPublication . Reis, Filipa Pereira; Teixeira, Alexandre; Cruz, David; Morgado, Ana; Arraiano, Cecília Maria; Romão, LuísaThe exosome is an evolutionarily conserved protein complex that is involved in all aspects of RNA metabolism, namely, RNA decay, processing and quality control. The only catalytic subunit of the core exosome is a 3´end exoribonuclease from the RNase II family of enzymes. In humans, two different homologues of this protein were identified, Dis3 and Dis3L1. While Dis3 mainly localizes in the nucleoplasm and has endonucleolytic activity, Dis3L1 is strictly cytoplasmic and has no endonucleolytic activity. The rapid decay of aberrant transcripts is not completely understood, but it is known that involves both 5’ to 3’ and 3’ to 5’ degradation. Despite that it localizes in the same compartment where NMD generally occurs, nothing is known about the role of Dis3L1 in quality control processes. In this work, we assessed the involvement of Dis3L1 in the 3’ to 5’ degradation of reporter human b-globin transcripts with premature termination and nonstop codons.
- Nonsense-mediated decay resistance of AUG-proximal nonsense-mutated transcripts relies on the interaction of PABPC1 with the translation initiation complexPublication . Peixeiro, Isabel; Barbosa, Cristina; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing a premature termination codon (PTC). The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmatic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail and associated PABPC1, in mRNAs containing downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human b-globin mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. We have reported that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. In fact, we have shown that the establishment of an efficient translation termination reaction at the AUG-proximal PTC is dependent on PABPC1 interaction with the initiation factor eIF4G and with the release factor eRF3 at the terminating ribosome. These interactions underlie critical 3’-5’ linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation. Furthermore, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. This work corroborates a role for PABPC1 on NMD evasion of transcripts carrying an AUG-proximal PTC and provides further insights into the mechanistic details of PTC definition and translation initiation.
- How far ‘AUG-proximity effect’ goes?Publication . Pereira, Francisco J.C.; Teixeira, Alexandre; Kong, Jian; Silva, Ana Luísa; Liebhaber, Stephen A.; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). We have previously shown that mRNAs carrying a PTC located in close proximity to the translation initiation AUG codon escape NMD. This was called the “AUG-proximity effect”. The present work illustrates that the extension of the AUG-proximity effect, i.e. to what position in the open reading frame (ORF) an AUG-proximal PTC does not trigger NMD, is different between human a- and b-globin mRNAs. Remarkably, our data also demonstrate that, contrary to what occurs in the b-globin transcripts, a-globin mRNAs carrying an AUG-proximal PTC allow for efficient translation re-initiation, although it only partially explains their NMD resistance. In addition, our results reveal that in the a- and b-globin transcripts, the extension of the AUG-proximity effect is determined by the ORF sequence. Furthermore, we show how the mRNA secondary structure, which is affected by the ORF sequence, determines the AUG-proximity effect extension. Our data point out that the time taken to translate the short ORF, affected by its sequence and stability, besides being involved in modulating translation re-initiation, also plays an important role in establishing the extension of the AUG-proximity effect.