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

Abstract

The DExD/H-box RNA helicase DHX34 is a nonsense-mediated decay (NMD) factor that together with core NMD factors coregulates 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 suggest that alterations in the levels and/or activity of DHX34 could contribute to human disease.

Translation‐coupled mRNA quality control mechanisms

“…These mutations map to different domains of DHX34, and all germline variants identified in these families abrogated NMD activity (Rio‐Machin et al , 2020 ). DHX34 is also associated with the human spliceosomal catalytic C complex and 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 (Hug et al , 2022 ).…”

Rapid UPF1 depletion illuminates the temporal dynamics of the NMD-regulated transcriptome in human cells

“…In contrast, both SMG6 and SMG7 are about 100 times less abundant and turn over more rapidly, explaining the faster onset of NMD inhibitory effects following their KD. To obtain a global view on NMD-inhibition, we investigated the transcriptome-wide effects of UPF1 KD across different human cell lines by analyzing recently published RNA-Seq datasets [74][75][76][77][78][79][80] . This strategy provides a broader perspective on UPF1 KD and aims to mitigate individual biases, since many of these datasets were generated in different laboratories using different KD protocols, target sequences, and cell lines.…”

“…Eleven publicly available RNA-Seq datasets were obtained and analyzed: I) UPF1 knockdown in HEK293 cells 77 (Gene Expression Omnibus (GEO) 143 accession GSE109143), II) UPF1 knockdown in HeLa cells 79 (Gene Expression Omnibus (GEO) accession GSE152435), III) UPF1 knockdown in HEK293 cells 74 (Gene Expression Omnibus (GEO) accession GSE176197), IV) UPF1 knockdown in K562 cells 76 (Gene Expression Omnibus (GEO) accession GSE204987), V) UPF1 knockdown in Huh7 cells 78 (Gene Expression Omnibus (GEO) accession GSE185655), VI) UPF1 knockdown in HCT116 cells 80 (Gene Expression Omnibus (GEO) accession GSE179843), VII) UPF1 knockdown in HepG2 cells 75 (Gene Expression Omnibus (GEO) accession GSE162199), VIII) SMG5, SMG6, SMG7 knockout/knockdown in HEK293 cells 19 (BioStudies 144,145 accession E-MTAB-9330), IX) UPF3A/B double knockout in HEK293 cells 81 (BioStudies accession E-MTAB-10716), X) CASC3 knockout in HEK293 cells 110 (BioStudies accession E-MTAB-8461) and XI) STAU1 knockout in HCT116 cells 102 For dataset #1, total RNA was extracted by lysing cells with TRIzol Reagent (Invitrogen). Libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) and sequenced on a NovaSeq6000 device (Illumina).…”

Synthetic lethal interactions of DEAD/H-box helicases as targets for cancer therapy

“…To disrupt nucleic acid structures, DEAD-box helicases use simple cycles of RNA duplex binding, unwinding, and release, while DEAH-box helicases function only as translocases in the 3’→5’ direction ( 17 ). In terms of their nucleic acid-related functions, both DEAD-box and DEAH-box proteins are implicated in virtually every aspect of RNA metabolic processes, including transcription ( 18 – 20 ), ribosomes biogenesis ( 21 , 22 ), small RNA process ( 23 , 24 ), pre-mRNA splicing ( 25 , 26 ), RNA storage and decay ( 27 , 28 ), nuclear export ( 29 – 31 ), liquid–liquid phase separation ( 32 – 34 ), RNA degradation ( 35 , 36 ), translation ( 37 – 42 ), and so on ( Figure 1B ). Some of them are also involved in DNA metabolism, such as DNA repair ( 43 – 47 ).…”