Identification of a localized nonsense-mediated decay pathway at the endoplasmic reticulum.

Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the endoplasmic reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Furthermore, we show that NBAS fulfills an independent function in NMD. This ER-NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 coregulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER-protective function by ensuring quality control of ER-translated mRNAs.

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes.

The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.

Slow RNAPII Transcription Elongation Rate, Low Levels of RNAPII Pausing, and Elevated Histone H1 Content at Promoters Associate with Higher m6A Deposition on Nascent mRNAs.

N6-methyladenosine modification (m6A) fine-tunes RNA fate in a variety of ways, thus regulating multiple fundamental biological processes. m6A writers bind to chromatin and interact with RNA polymerase II (RNAPII) during transcription. To evaluate how the dynamics of the transcription process impact m6A deposition, we studied RNAPII elongation rates in mouse embryonic stem cells with altered chromatin configurations, due to reductions in linker histone H1 content. We found that genes transcribed at slow speed are preferentially methylated and display unique signatures at their promoter region, namely high levels of histone H1, together with marks of bivalent chromatin and low RNAPII pausing. They are also highly susceptible to m6A loss upon histone H1 reduction. These results indicate that RNAPII velocity links chromatin structure and the deposition of m6A, highlighting the intricate relationship between different regulatory layers on nascent mRNA molecules.

Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner.

Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we identify a function of histone H1 in chromatin structure and transcription using a range of genomic approaches. In the absence of histone H1, there is an increase in the transcription of non-coding RNAs, together with reduced levels of m6A modification leading to their accumulation on chromatin and causing replication-transcription conflicts. This strongly suggests that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism, and DNA replication to maintain genome performance.

Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function.

Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus, and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells and tissues derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

The translational landscape of the splicing factor SRSF1 and its role in mitosis.

The shuttling Serine/Arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1,500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets; strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer.

Anterior Gradient-3: a novel biomarker for ovarian cancer that mediates cisplatin resistance in xenograft models.

The Anterior Gradient (AGR) genes AGR2 and AGR3 are part of the Protein Disulfide Isomerase (PDI) family and harbour core thioredoxin folds (CxxS motifs) that have the potential to regulate protein folding and maturation. A number of proteomics and transcriptomics screens in the fields of limb regeneration, cancer cell metastasis, pro-oncogenic oestrogen-signalling, and p53 regulation have identified AGR2 as a novel component of these signalling pathways. Curiously, despite the fact that the AGR2 and AGR3 genes are contiguous on chromosome 7p21.1-3, the AGR3 protein has rarely been identified in such OMICs screens along with AGR2 protein. Therefore there is little information on how AGR3 protein is expressed in normal and diseased states. A panel of three monoclonal antibodies was generated towards AGR3 protein for identifying novel clinical models that can be used to define whether AGR3 protein could play a positive or negative role in human cancer development. One monoclonal antibody was AGR3-specific and bound a linear epitope that could be defined using both pep-scan and phage-peptide library screening. Using this monoclonal antibody, endogenous AGR3 protein expression was shown to be cytosolic in four human ovarian cancer subtypes; serous, endometrioid, clear cell, and mucinous. Mucinous ovarian cancers produced the highest number of AGR3 positive cells. AGR3 expression is coupled to AGR2 expression only in mucinous ovarian cancers, whereas AGR3 and AGR2 expressions are uncoupled in the other three types of ovarian cancer. AGR3 expression in ovarian cancer is independent of oestrogen-receptor expression, which is distinct from the oestrogen-receptor dependent expression of AGR3 in breast cancers. Isogenic cancer cell models were created that over-express AGR3 and these demonstrated that AGR3 mediates cisplatin-resistance in mouse xenografts. These data indicate that AGR3 is over-expressed by a hormone (oestrogen-receptor α)-independent mechanism and identify a novel protein-folding associated pathway that could mediate resistance to DNA-damaging agents in human cancers.

A divergent substrate-binding loop within the pro-oncogenic protein anterior gradient-2 forms a docking site for Reptin.

Anterior gradient-2 (AGR2) functions in a range of biological systems, including goblet cell formation, limb regeneration, inhibition of p53, and metastasis. There are no well-validated binding proteins for AGR2 protein despite the wealth of data implicating an important cellular function in vertebrates. The yeast two-hybrid system was used to isolate the ATP binding protein Reptin as an AGR2-interacting protein. AGR2 formed a stable complex in human cell lysates with Reptin, thus validating Reptin as an AGR2 binding protein in cells. Reptin was also shown to be overproduced in a panel of primary breast cancer biopsy specimens, relative to normal adjacent tissue from the same patient, suggesting a role in cancer growth in vivo. Mutations were made at the two ATP binding motifs in Reptin to evaluate the effects of ATP on Reptin-AGR2 complex stability. Loss-of-ATP binding mutations at the Walker A motif (K83A) or gain-of-ATP binding mutations at the Walker B motif (D299N) resulted in Reptin mutants with altered oligomerization, thermostability, and AGR2 binding properties. These data indicate that the two ATP binding motifs of Reptin play a role in regulating the stability of the AGR2-Reptin complex. The minimal region of AGR2 interacting with Reptin was localized using overlapping peptide libraries derived from the AGR2 protein sequence. The Reptin docking site was mapped to a divergent octapeptide loop in the AGR2 superfamily between amino acids 104 and 111. Mutations at codon Y104 or F111 in full-length AGR2 destabilized the binding of Reptin. These data highlight the existence of a protein docking motif on AGR2 and an ATP-regulated peptide-binding activity for Reptin. This knowledge has implications for isolating other AGR2-interacting proteins, for developing assays to isolate small molecules that target the Reptin ATP binding site, and for measuring the effects of the Reptin-AGR2 complex in cancer cell growth.