Biology subjects

Caceres, J. F.

Publications and source records attributed to Caceres, J. F..

8 recordsLinked to original sources

bioRxiv2026

The moonlighting functions of SEC13 in nonsense-mediated mRNA decay and the regulation of ER stress

The nonsense-mediated decay pathway (NMD) is an RNA quality control mechanism that regulates the stability of target RNAs. We previously identified the ER-localized SEC13 protein as a novel NMD factor in C. elegans and in HeLa cells; raising the possibility that it could be involved in regulating the stability of mRNAs translated at the ER. SEC13 is a component of several cellular complexes, including the COPII vesicle coat, the nuclear pore complex (NPC) and the nutrient sensing GATOR2 complex. Here, we show that SEC13 interacts with core NMD factors and using a newly developed dual-color fluorescent NMD sensor in U2OS cells, we assessed SEC13 NMD activity, at a single-cell level. Transcriptomic profiling revealed that unlike the previously described ER-NMD factor, NBAS, SEC13 co-regulates the stability of substrates translated both in the cytoplasm and at the ER. We also show that SEC13 function in NMD is largely independent of its function in other cellular complexes. Altogether, these results show that SEC13 is a bona fide NMD factor in mammalian cells. Finally, we utilized an ER stress-activated indicator (ERAI) in U2OS cells to demonstrate that SEC13, together with canonical NMD factors, has a role in the regulation of the unfolded protein response (UPR) at the ER. Thus, the moonlighting functions of SEC13 include a role in NMD pathway and the regulation of ER stress.

molecular biology↗

bioRxiv2022

A dual role for the RNA helicase DHX34 in NMDand pre-mRNA splicing and its function inhematopoietic differentiation

The DExD/H-box RNA helicase DHX34 is a Nonsense-mediated decay (NMD) factor that together with core NMD factors co-regulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using Cross-Linking Immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon-intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial Myelodysplasia (MDS)/Acute Myeloid Leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggests that alterations in the levels and/or activity of DHX34 could contribute to human disease.

molecular biology↗

bioRxiv2021

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 have identified a new function of histone H1 in chromatin structure and transcription using a range of genomic approaches. We show that histone H1-depleted cells accumulate nascent non-coding RNAs on chromatin, suggesting that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accumulated non-coding transcripts have reduced levels of m6A modification and cause replication-transcription conflicts. 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.

molecular biology↗

bioRxiv2020

Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1 and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

biophysics↗

bioRxiv2020

Translational activity of the 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 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.

molecular biology↗

bioRxiv2020

Identification of a 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. Here, we show that NBAS fulfils 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 co-regulate 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/954453v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@172e4a3org.highwire.dtl.DTLVardef@9769org.highwire.dtl.DTLVardef@d478c1org.highwire.dtl.DTLVardef@143e0f9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LINBAS is an NMD factor that localizes to the membrane of the endoplasmic reticulum (ER) C_LIO_LINBAS has dual, independent, roles in Golgi-to-ER retrograde transport and in ER-NMD C_LIO_LINBAS recruits the core NMD factor UPF1 to the membrane of the ER C_LIO_LIThe ER-NMD pathway targets for degradation mRNAs that are translated at the ER C_LI

molecular biology↗

bioRxiv2018

Slow transcriptional elongation causes embryonic lethality and perturbs kinetic coupling of long neural genes

Alternative splicing (AS) is a highly regulated process that increases protein diversity and is critical for cell differentiation and development. The rate of RNA Polymerase II (RNAPII) elongation has an important role in the control of AS. We generated mouse embryonic stem cells (ESCs) knocked-in for a slow elongating form of RNAPII and show that a reduced transcriptional elongation rate causes early embryonic lethality in mice and impairs the differentiation of ESCs into the neural lineage. The reduced elongation rate caused changes in splicing and in gene expression in ESCs and along the pathway of neuronal differentiation. 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 more predominant 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.

molecular biology↗

bioRxiv2016

Genetic variation and RNA structure regulate microRNA biogenesis

MiRNA biogenesis is highly regulated at the post-transcriptional level; however, the role of sequence and secondary RNA structure in this process has not been extensively studied. A single G to A substitution present in the terminal loop of pri-mir-30c-1 in breast cancer patients had been previously described to result in increased levels of mature miRNA. Here, we report that this genetic variant directly affects Drosha-mediated processing of pri-mir-30c-1 in vitro and in cultured cells. Structural analysis of this variant revealed an altered RNA structure that facilitates the interaction with SRSF3, an SR protein family member that promotes pri-miRNA processing. Our results are compatible with a model whereby a genetic variant in pri-mir-30c-1 leads to a secondary RNA structure rearrangement that facilitates binding of SRSF3 resulting in increased levels of miR-30c. These data highlights that primary sequence determinants and RNA structure are key regulators of miRNA biogenesis.

biochemistry↗