Coupled protein quality control during nonsense mediated mRNA decay

Translation of mRNAs containing premature termination codons (PTCs) can result in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance path-way responsible for detecting and degrading PTC containing transcripts. While the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational, and dependent on an intact ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors, and suggested a lack of dependence on the canonical ribosome-quality control (RQC) pathway. Finally, one of the strongest hits in our screens was the E3 ubiquitin ligase CNOT4, a member of the CCR4-NOT complex, which is involved in initiating mRNA degradation. We show that CNOT4 is involved in NMD coupled protein degradation, and its role depends on a functional RING ubiquitin ligase domain. Our results demonstrate the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a framework for identifying and characterizing factors involved in this process.

Nonsense-mediated mRNA decay uses complementary mechanisms to suppress mRNA and protein accumulation

Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons. Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to eightfold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.

Cellular variability of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two β-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.

RNF219 attenuates global mRNA decay through inhibition of CCR4-NOT complex-mediated deadenylation

The CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon HDAC inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified RNF219 as an acetylation-regulated cofactor. We demonstrate that RNF219 is an active RING-type E3 ligase which stably associates with CCR4-NOT via NOT9 through a short linear motif (SLiM) embedded within the C-terminal low-complexity region of RNF219. By using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 inhibits deadenylation through the direct interaction of the α-helical SLiM with the NOT9 module. Transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, with differential requirement of its RING domain. Our results establish RNF219 as an inhibitor of CCR4-NOT-mediated deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover.

Ribosomal ubiquitination facilitates mRNA cleavage and ribosome rescue during No-Go and Nonstop mRNA Decay

During translational surveillance, ribosomes play a critical role in detecting problematic mRNAs and signaling cellular machinery to repress the offending messages. Prior work has shown that problematic mRNAs identified by two surveillance pathways (Nonstop and No-Go mRNA Decay) are detected by ribosome collisions and subsequent ribosomal ubiquitination, yet how ribosomal ubiquitination leads to repression has remained unclear. Here, we deploy C. elegans to unravel the series of coordinated events during Nonstop and No-Go mRNA Decay. We probe the metazoan SKI RNA helicase complex to uncover functionally significant residues and reveal divergence of the SKI-exosome interface. We define a functional requirement for ubiquitination on at least two ribosomal proteins during No-Go mRNA Decay, and illustrate how ubiquitination recruits the endonuclease NONU-1 via CUE domains and the ribosome rescue factor HBS-1 via its poorly characterized N-terminus. Our molecular characterization (1) underscores the importance of ribosomal ubiquitination in mRNA degradation, (2) shows similar and distinct genetic dependencies of factors in Nonstop and No-Go mRNA Decay, and (3) uncovers a conspicuous absence of distinct ribosomal stalls at No-Go mRNA Decay substrates. Our work demonstrates mechanisms by which translation signals to effectors of co-translational mRNA repression and has implications for the study of translation and ribosomal species in vivo.

PABPN1L assemble into “ring-like” aggregates in the cytoplasm of MII oocytes and is associated with female infertility†

Infertility affects 10% - 15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. A resent study showed that PABPN1L recruited BTG4 to mRNA 3′-poly(A) tails and was essential for maternal mRNA degradation. Here, we generated an PABPN1L-antibody and found “ring-like” PABPN1L aggregates in the cytoplasm of MII oocytes. PABPN1L-EGFP proteins spontaneously formed“ring-like” aggregates in vitro. This phenomenon is similar with CCR4–NOT catalytic subunit, CNOT7, when it starts deadenylation process in vitro. We constructed two mouse model (Pabpn1l −/− and Pabpn1l tm1a/tm1a) simulating the intron1-exon2 abnormality of human PABPN1L and found that the female was sterile and the male was fertile. Using RNA-Seq, we observed a large-scale up-regulation of RNA in zygotes derived from Pabpn1l−/− MII oocytes. We found that 9222 genes were up-regulated instead of being degraded in the Pabpn1l-♀/+♂zygote. Both the Btg4 and Cnot61 genes are necessary for the deadenylation process and Pabpn1l −/− resembled both the Btg4 and Cnot6l knockouts, where 71.2% genes stabilized in the Btg4-♀/+♂ zygote and 84.2% genes stabilized in the Cnot6l-♀/+♂zygote were also stabilized in Pabpn1l-♀/+♂ zygote. BTG4/CNOT7/CNOT6L was partially co-located with PABPN1L in MII oocytes. The above results suggest that PABPN1L is widely associated with CCR4–NOT-mediated maternal mRNA degradation and PABPN1L variants on intron1-exon2 could be a genetic marker of female infertility. Summary sentence. “Ring-like” PABPN1L aggregates was found in the cytoplasm of MII oocytes and in vitro; intron1-exon2 abnormality of Pabpn1l leads female sterile in mice.

Polyribosome-Dependent Clustering of Membrane-Anchored RNA Degradosomes To Form Sites of mRNA Degradation in Escherichia coli

Here, we show that RNase E, RhlB, and PNPase act together as components of the multienzyme RNA degradosome in polyribosome-dependent clustering to form puncta on the inner cytoplasmic membrane. Our results support the hypothesis that RNA degradosome puncta are sites of mRNA degradation.