The G-patch protein NF-κB-repressing factor mediates the recruitment of the exonuclease XRN2 and activation of the RNA helicase DHX15 in human ribosome biogenesis

Abstract

In eukaryotes, the synthesis of ribosomal subunits, which involves the maturation of the ribosomal (r)RNAs and assembly of ribosomal proteins, requires the co-ordinated action of a plethora of ribosome biogenesis factors. Many of these cofactors remain to be characterized in human cells. Here, we demonstrate that the human G-patch protein NF-κB-repressing factor (NKRF) forms a pre-ribosomal subcomplex with the DEAH-box RNA helicase DHX15 and the 5΄-3΄ exonuclease XRN2. Using UV crosslinking and analysis of cDNA (CRAC), we reveal that NKRF binds to the transcribed spacer regions of the pre-rRNA transcript. Consistent with this, we find that depletion of NKRF, XRN2 or DHX15 impairs an early pre-rRNA cleavage step (A’). The catalytic activity of DHX15, which we demonstrate is stimulated by NKRF functioning as a cofactor, is required for efficient A’ cleavage, suggesting that a structural remodelling event may facilitate processing at this site. In addition, we show that depletion of NKRF or XRN2 also leads to the accumulation of excised pre-rRNA spacer fragments and that NKRF is essential for recruitment of the exonuclease to nucleolar pre-ribosomal complexes. Our findings therefore reveal a novel pre-ribosomal subcomplex that plays distinct roles in the processing of pre-rRNAs and the turnover of excised spacer fragments.

Comparison of preribosomal RNA processing pathways in yeast, plant and human cells – focus on coordinated action of endo‐ and exoribonucleases

“…), however, the functional importance of this mechanism is not fully determined, as lack or delay in P cleavage in plants or 01(A′) in mammals causes minor changes in the downstream processing steps and does not affect production of mature rRNAs . Importantly, human XRN2 has been very recently shown to act as a component of the novel nucleolar subcomplex, together with the NKRF/NFκB factor and a DEAH‐box helicase DHX15, a homologue of the yeast Prp43 that facilitates Nob1‐mediated cleavage (Table S1; see section PIN domain‐containing proteins, part about Nob1). Both the DHX15 and NKRF are required for the 01(A′) cleavage to occur – NKRF recruits XRN2 and stimulates DHX15 enzymatic activity, which likely induces structural rearrangement of the substrate around processing site 01(A′) .…”

“…It was shown in plants that the exonucleolytic trimming of the 5′‐ETS by AtXRN2/3 facilitates the P cleavage to occur , but in humans the exact mechanism of XRN2 activity on pre‐rRNA remains elusive . It was proposed, however, that the 01(A′) cleavage is facilitated by the remodelling of pre‐rRNA by the DHX15 helicase, which is in turn stimulated by the NKRF factor that associates also with XRN2, and interactions between these three proteins are crucial for efficient assembly of human ribosomes, at least at the early stages, and particularly in response to stress‐producing stimuli . Human XRN2 likely plays a more universal role as a quality controller and coordinator of the proper sequence of events during pre‐rRNA processing .…”

Diverse Roles of DEAD/DEAH-Box Helicases in Innate Immunity and Diseases

“…RBM5 is an alternative splicing regulator of apoptosis-related genes, thus implying that DHX15 regulates apoptosis through splicing of these genes [99]. In nucleolus, DHX15 participates in ribosome biogenesis, forming a complex with NKRF (NF-κB-repressing factor) and exonuclease XRN2 [100]. Apart from these functions, DHX15 located in cytosol and on mitochondrial membrane plays a role in in antiviral innate immune response and signaling [96À98].…”

“…These differences suggest existence of interaction between DHX15 and cell/tissue type-specific proteins which modulate the activity of DHX15 and MAVS signaling pathway. Moreover, the fact that the nucleolar DHX15 interacts with NKRF points out to the possibility that this DEAH-box helicase may participate in NF-κB signaling on two different levels [100,154,155]. Further research is needed to gain better understanding of the activities of DHX15.…”

“…DHX15 is divided into six structural domains, namely the N-terminal extension, the RecA1 and RecA2 domains, the winged-helix domain, the ratchet domain and the oligonucleotide /oligosaccharidebinding (OB)-fold domain. Among them, the OB-fold domain is important for interaction with the G-patch proteins, such as NKRF [100,156].…”