SUZ domain-containing proteins have multiple effects on nonsense-mediated decay target transcripts.

Many transcripts are targeted by nonsense-mediated decay (NMD), leading to their degradation and the inhibition of their translation. We found that the protein SUZ domain-containing protein 1 (SZRD1) interacts with the key NMD factor up-frameshift 1. When recruited to NMD-sensitive reporter gene transcripts, SZRD1 increased protein production, at least in part, by relieving translational inhibition. The conserved SUZ domain in SZRD1 was required for this effect. The SUZ domain is present in only three other human proteins besides SZRD1: R3H domain-containing protein 1 and 2 (R3HDM1, R3HDM2) and cAMP-regulated phosphoprotein 21 (ARPP21). We found that ARPP21, similarly to SZRD1, can increase protein production from NMD-sensitive reporter transcripts in an SUZ domain-dependent manner. This indicated that the SUZ domain-containing proteins could prevent translational inhibition of transcripts targeted by NMD. Consistent with the idea that SZRD1 mainly prevents translational inhibition, we did not observe a systematic decrease in the abundance of NMD targets when we knocked down SZRD1. Surprisingly, knockdown of SZRD1 in two different cell lines led to reduced levels of the NMD component UPF3B, which was accompanied by increased levels in a subset of NMD targets. This suggests that SZRD1 is required to maintain normal UPF3B levels and indicates that the effect of SZRD1 on NMD targets is not limited to a relief from translational inhibition. Overall, our study reveals that human SUZ domain-containing proteins play a complex role in regulating protein output from transcripts targeted by NMD.

Extracellular vesicles from endothelial progenitor cells promote thyroid follicle formation.

Organogenesis is a complex and dynamic process requiring reciprocal communication between different cell types. In the thyroid, thyrocyte progenitors secrete the angiocrine factor, VEGFA, to recruit endothelial cells. In return, endothelial cells promote thyrocyte organisation into spherical follicular structures, which are responsible for thyroid hormone synthesis and storage. Medium conditioned by endothelial progenitor cells (EPCs) can promote follicle formation and lumen expansion (i.e. folliculogenesis) in an ex vivo culture system of thyroid lobes. Here, we postulated that endothelial cells instruct thyrocyte progenitors by producing extracellular vesicles (EVs). We found that medium conditioned by EPCs contain EVs with exosomal characteristics and that these vesicles can be incorporated into thyrocyte progenitors. By mass spectrometry, laminin peptides were abundantly identified in the EV preparations, probably co-sedimenting with EVs. Laminin-α1 silencing in EPC abrogated the folliculogenic effect of EVs. However, density gradient separation of EVs from laminins revealed that both EV-rich and laminin-rich fractions exhibited folliculogenic activity. In conclusion, we suggest that endothelial cells can produce EVs favouring thyrocyte organisation into follicles and lumen expansion, a mechanism promoted by laminin-α1.

ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto α-dystroglycan.

Mutations in genes required for the glycosylation of α-dystroglycan lead to muscle and brain diseases known as dystroglycanopathies. However, the precise structure and biogenesis of the assembled glycan are not completely understood. Here we report that three enzymes mutated in dystroglycanopathies can collaborate to attach ribitol phosphate onto α-dystroglycan. Specifically, we demonstrate that isoprenoid synthase domain-containing protein (ISPD) synthesizes CDP-ribitol, present in muscle, and that both recombinant fukutin (FKTN) and fukutin-related protein (FKRP) can transfer a ribitol phosphate group from CDP-ribitol to α-dystroglycan. We also show that ISPD and FKTN are essential for the incorporation of ribitol into α-dystroglycan in HEK293 cells. Glycosylation of α-dystroglycan in fibroblasts from patients with hypomorphic ISPD mutations is reduced. We observe that in some cases glycosylation can be partially restored by addition of ribitol to the culture medium, suggesting that dietary supplementation with ribitol should be evaluated as a therapy for patients with ISPD mutations.

KPC2 relocalizes HOXA2 to the cytoplasm and decreases its transcriptional activity.

Regulation of transcription factor activity relies on molecular interactions or enzymatic modifications which influence their interaction with DNA cis-regulatory sequences, their transcriptional activation or repression, and stability or intracellular distribution of these proteins. Regarding the well-conserved Hox protein family, a restricted number of activity regulators have been highlighted thus far. In the framework of a proteome-wide screening aiming at identifying proteins interacting with Hoxa2, KPC2, an adapter protein constitutive of the KPC ubiquitin-ligase complex, was identified. In this work, KPC2 was confirmed as being a genuine interactor of Hoxa2 by co-precipitation and bimolecular fluorescence complementation assays. At functional level, KPC2 diminishes the transcriptional activity and induces the nuclear exit of Hoxa2. Gene expression analyses revealed that Kpc2 is active in restricted areas of the developing mouse embryo which overlap with the Hoxa2 expression domain. Together, our data support that KPC2 regulates Hoxa2 by promoting its relocation to the cytoplasm.

A unique C-terminal domain allows retention of matrix metalloproteinase-27 in the endoplasmic reticulum.

Matrix metalloproteinase-27 (MMP-27) is poorly characterized. Sequence comparison suggests that a C-terminal extension (CTE) includes a potential transmembrane domain as in some membrane-type (MT)-MMPs. Having noticed that MMP-27 was barely secreted, we investigated its subcellular localization and addressed CTE contribution for MMP-27 retention. Intracellular MMP-27 was sensitive to endoglycosidase H. Subcellular fractionation and confocal microscopy evidenced retention of endogenous MMP-27 or recombinant rMMP-27 in the endoplasmic reticulum (ER) with locked exit across the intermediate compartment (ERGIC). Conversely, truncated rMMP-27 without CTE accessed downstream secretory compartments (ERGIC and Golgi) and was constitutively secreted. CTE addition to rMMP-10 (a secreted MMP) caused ER retention and blocked secretion. Addition of a PKA target sequence to the cytosolic C-terminus of transmembrane MT1-MMP/MMP-14 led to effective phosphorylation upon forskolin stimulation, but not for MMP-27, excluding transmembrane anchorage. Moreover, MMP-27 was protected from digestion by proteinase K. Finally, MT1-MMP/MMP-14 but neither endogenous nor recombinant MMP-27 partitioned in the detergent phase after Triton X-114 extraction, indicating that MMP-27 is not an integral membrane protein. In conclusion, MMP-27 is efficiently retained within the ER due to its unique CTE, which does not lead to stable membrane insertion. This could represent a novel ER retention system.