Ribosome biogenesis requires multiple nuclease activities to process pre-rRNA transcripts into mature rRNA species and eliminate defective products of transcription and processing. We find that in mammalian cells, the 5′ exonuclease Xrn2 plays a major role in both maturation of rRNA and degradation of a variety of discarded pre-rRNA species. Precursors of 5.8S and 28S rRNAs containing 5′ extensions accumulate in mouse cells after siRNA-mediated knockdown of Xrn2, indicating similarity in the 5′-end maturation mechanisms between mammals and yeast. Strikingly, degradation of many aberrant pre-rRNA species, attributed mainly to 3′ exonucleases in yeast studies, occurs 5′ to 3′ in mammalian cells and is mediated by Xrn2. Furthermore, depletion of Xrn2 reveals pre-rRNAs derived by cleavage events that deviate from the main processing pathway. We propose that probing of pre-rRNA maturation intermediates by exonucleases serves the dual function of generating mature rRNAs and suppressing suboptimal processing paths during ribosome assembly.

Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.

Ribosome biogenesis is a dynamic multistep process, many features of which are still incompletely documented. Here, we show that changes in this pathway can be captured and annotated by means of a graphic set of pre-rRNA ratios, a technique we call Ratio Analysis of Multiple Precursors (RAMP). We find that knocking down a ribosome synthesis factor produces a characteristic RAMP profile that exhibits consistency across a range of depletion levels. This facilitates the inference of affected steps and simplifies comparative analysis. We applied RAMP to examine how endonucleolytic cleavages of the mouse pre-rRNA transcript in the internal transcribed spacer 1 (ITS1) are affected by depletion of factors required for maturation of the small ribosomal subunit (Rcl1, Fcf1/Utp24, Utp23) and the large subunit (Pes1, Nog1). The data suggest that completion of early maturation in a subunit triggers its release from the common pre-rRNA transcript by stimulating cleavage at the proximal site in ITS1. We also find that splitting of pre-rRNA in the 3′ region of ITS1 is prevalent in adult mouse tissues and quiescent cells, as it is in human cells. We propose a model for subunit separation during mammalian ribosome synthesis and discuss its implications for understanding pre-rRNA processing pathways.

Regulation of the actin cytoskeleton is crucial for normal development and function of the immune system, as evidenced by the severe immune abnormalities exhibited by patients bearing inactivating mutations in the Wiskott Aldrich Syndrome Protein (WASP), a key regulator of actin dynamics. WASP exerts its effects on actin dynamics through a multi-subunit complex termed Arp2/3. Despite the critical role played by Arp2/3 as an effector of WASP-mediated control over actin polymerization, mutations in protein components of the Arp2/3 complex had not previously been identified as a cause of immunodeficiency. Here, we describe two brothers with hematopoietic and immunologic symptoms reminiscent of Wiskott Aldrich Syndrome (WAS). However, these patients lacked mutations in any of the genes previously associated with WAS. Whole exome sequencing (WES) revealed a homozygous 2 bp deletion, n.c.G623DEL-TC (p.V208fs), in Arp2/3 complex component ARPC1B, that causes a frame shift resulting in premature termination. Modeling of the disease in zebrafish revealed that ARPC1B plays a critical role in supporting T cell and thrombocyte development. Moreover, the defects in development caused by ARPC1B loss could be rescued by the intact human ARPC1B ortholog, but not by the p.V208fs variant identified in the patient. Moreover, we found that the expression of ARPC1B is restricted to hematopoietic cells, potentially explaining why a mutation in ARPC1B has now been observed as a cause of WAS, while mutations in other, more widely expressed, components of the Arp2/3 complex have not been observed.

SUMMARY Most ribosomal proteins (RP) are regarded as essential, static components that only contribute to ribosome biogenesis and protein synthesis. However, emerging evidence suggests that RNA-binding RP are dynamic and can influence cellular processes by performing “extraribosomal”, regulatory functions involving binding to select, critical target mRNAs. We report here that the RP, Rpl22, and its highly homologous paralog, Rpl22-Like1 (Rpl22l1 or Like1), play critical, extraribosomal roles in embryogenesis. Indeed, they antagonistically control morphogenesis through developmentally-regulated localization to the nucleus where they modulate splicing of the pre-mRNA encoding smad2, an essential transcriptional effector of Nodal/TGF-β signaling. During gastrulation, Rpl22 binds to intronic sequences of smad2 pre-mRNA and induces exon 9 skipping in cooperation with hnRNP-A1. This action is opposed by its paralog, Like1, which promotes exon 9 inclusion in the mature transcript. The nuclear roles of these RP in controlling morphogenesis represent a fundamentally different and paradigm-shifting mode of action for RP.