Spliceosome Profiling Visualizes Operations of a Dynamic RNP at Nucleotide Resolution.

Tools to understand how the spliceosome functions in vivo have lagged behind advances in the structural biology of the spliceosome. Here, methods are described to globally profile spliceosome-bound pre-mRNA, intermediates, and spliced mRNA at nucleotide resolution. These tools are applied to three yeast species that span 600 million years of evolution. The sensitivity of the approach enables the detection of canonical and non-canonical events, including interrupted, recursive, and nested splicing. This application of statistical modeling uncovers independent roles for the size and position of the intron and the number of introns per transcript in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal the ATP-dependent discard of numerous endogenous substrates after spliceosome assembly in vivo and connect this discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology used to investigate an RNP central to eukaryotic gene expression.

A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal.

Product binding enforces the genomic specificity of a yeast polycomb repressive complex.

We characterize the Polycomb system that assembles repressive subtelomeric domains of H3K27 methylation (H3K27me) in the yeast Cryptococcus neoformans. Purification of this PRC2-like protein complex reveals orthologs of animal PRC2 components as well as a chromodomain-containing subunit, Ccc1, which recognizes H3K27me. Whereas removal of either the EZH or EED ortholog eliminates H3K27me, disruption of mark recognition by Ccc1 causes H3K27me to redistribute. Strikingly, the resulting pattern of H3K27me coincides with domains of heterochromatin marked by H3K9me. Indeed, additional removal of the C. neoformans H3K9 methyltransferase Clr4 results in loss of both H3K9me and the redistributed H3K27me marks. These findings indicate that the anchoring of a chromatin-modifying complex to its product suppresses its attraction to a different chromatin type, explaining how enzymes that act on histones, which often harbor product recognition modules, may deposit distinct chromatin domains despite sharing a highly abundant and largely identical substrate-the nucleosome.

Primate-specific ORF0 contributes to retrotransposon-mediated diversity.

LINE-1 retrotransposons are fast-evolving mobile genetic entities that play roles in gene regulation, pathological conditions, and evolution. Here, we show that the primate LINE-1 5'UTR contains a primate-specific open reading frame (ORF) in the antisense orientation that we named ORF0. The gene product of this ORF localizes to promyelocytic leukemia-adjacent nuclear bodies. ORF0 is present in more than 3,000 loci across human and chimpanzee genomes and has a promoter and a conserved strong Kozak sequence that supports translation. By virtue of containing two splice donor sites, ORF0 can also form fusion proteins with proximal exons. ORF0 transcripts are readily detected in induced pluripotent stem (iPS) cells from both primate species. Capped and polyadenylated ORF0 mRNAs are present in the cytoplasm, and endogenous ORF0 peptides are identified upon proteomic analysis. Finally, ORF0 enhances LINE-1 mobility. Taken together, these results suggest a role for ORF0 in retrotransposon-mediated diversity.

The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans.

Eukaryotic cells regulate 5'-triphosphorylated RNAs (ppp-RNAs) to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR-1 (phosphatase that interacts with RNA and ribonucleoprotein particle 1) family of RNA polyphosphatases remove both the ß and γ phosphates from ppp-RNAs. Here, we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RNA-dependent RNA polymerases (RdRPs) and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and Dicer-independent Argonaute pathways and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.

Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans.

During each life cycle, germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here, we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small RNA signal that represents an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together, these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small RNA complexes that constitute a memory of gene expression in preceding generations.

Stalled spliceosomes are a signal for RNAi-mediated genome defense.

Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of expression signals encoded by transposons versus host genes offers an avenue for the evolution of genome defense.

A tick saliva serpin, IxsS17 inhibits host innate immune system proteases and enhances host colonization by Lyme disease agent.

Lyme disease (LD) caused by Borrelia burgdorferi is among the most important human vector borne diseases for which there is no effective prevention method. Identification of tick saliva transmission factors of the LD agent is needed before the highly advocated tick antigen-based vaccine could be developed. We previously reported the highly conserved Ixodes scapularis (Ixs) tick saliva serpin (S) 17 (IxsS17) was highly secreted by B. burgdorferi infected nymphs. Here, we show that IxsS17 promote tick feeding and enhances B. burgdorferi colonization of the host. We show that IxsS17 is not part of a redundant system, and its functional domain reactive center loop (RCL) is 100% conserved in all tick species. Yeast expressed recombinant (r) IxsS17 inhibits effector proteases of inflammation, blood clotting, and complement innate immune systems. Interestingly, differential precipitation analysis revealed novel functional insights that IxsS17 interacts with both effector proteases and regulatory protease inhibitors. For instance, rIxsS17 interacted with blood clotting proteases, fXII, fX, fXII, plasmin, and plasma kallikrein alongside blood clotting regulatory serpins (antithrombin III and heparin cofactor II). Similarly, rIxsS17 interacted with both complement system serine proteases, C1s, C2, and factor I and the regulatory serpin, plasma protease C1 inhibitor. Consistently, we validated that rIxsS17 dose dependently blocked deposition of the complement membrane attack complex via the lectin complement pathway and protected complement sensitive B. burgdorferi from complement-mediated killing. Likewise, co-inoculating C3H/HeN mice with rIxsS17 and B. burgdorferi significantly enhanced colonization of mouse heart and skin organs in a reverse dose dependent manner. Taken together, our data suggests an important role for IxsS17 in tick feeding and B. burgdorferi colonization of the host.

Essential requirement for IER3IP1 in B cell development.

In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for aberrant immune function, we identified animals with low percentages of B220+ cells in the peripheral blood. The causative mutation was in Ier3ip1, encoding immediate early response 3 interacting protein 1 (IER3IP1), an endoplasmic reticulum membrane protein mutated in an autosomal recessive neurodevelopmental disorder termed Microcephaly with simplified gyration, Epilepsy and permanent neonatal Diabetes Syndrome (MEDS) in humans. However, no immune function for IER3IP1 had previously been reported. The viable hypomorphic Ier3ip1 allele uncovered in this study, identical to a reported IER3IP1 variant in a MEDS patient, reveals an essential hematopoietic-intrinsic role for IER3IP1 in B cell development and function. We show that IER3IP1 forms a complex with the Golgi transmembrane protein 167A and limits activation of the unfolded protein response mediated by inositol-requiring enzyme-1α and X-box binding protein 1 in B cells. Our findings suggest that B cell deficiency may be a feature of MEDS.

Essential role of MFSD1-GLMP-GIMAP5 in lymphocyte survival and liver homeostasis.

We detected ENU-induced alleles of Mfsd1 (encoding the major facilitator superfamily domain containing 1 protein) that caused lymphopenia, splenomegaly, progressive liver pathology, and extramedullary hematopoiesis (EMH). MFSD1 is a lysosomal membrane-bound solute carrier protein with no previously described function in immunity. By proteomic analysis, we identified association between MFSD1 and both GLMP (glycosylated lysosomal membrane protein) and GIMAP5 (GTPase of immunity-associated protein 5). Germline knockout alleles of Mfsd1, Glmp, and Gimap5 each caused lymphopenia, liver pathology, EMH, and lipid deposition in the bone marrow and liver. We found that the interactions of MFSD1 and GLMP with GIMAP5 are essential to maintain normal GIMAP5 expression, which in turn is critical to support lymphocyte development and liver homeostasis that suppresses EMH. These findings identify the protein complex MFSD1-GLMP-GIMAP5 operating in hematopoietic and extrahematopoietic tissues to regulate immunity and liver homeostasis.

Insulin secretion deficits in a Prader-Willi syndrome ß-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones.

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in ß-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS ß-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS ß-cells. Consistent with reduced ER chaperones levels, PWS INS-1 ß-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS ß-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic ß-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and ß-cell secretory pathway function.

RNPS1 inhibits excessive tumor necrosis factor/tumor necrosis factor receptor signaling to support hematopoiesis in mice.

Null mutations of spliceosome components or cofactors are homozygous lethal in eukaryotes, but viable hypomorphic mutations provide an opportunity to understand the physiological impact of individual splicing proteins. We describe a viable missense allele (F181I) of Rnps1 encoding an essential regulator of splicing and nonsense-mediated decay (NMD), identified in a mouse genetic screen for altered immune cell development. Homozygous mice displayed a stem cell­intrinsic defect in hematopoiesis of all lineages due to excessive apoptosis induced by tumor necrosis factor (TNF)­dependent death signaling. Numerous transcript splice variants containing retained introns and skipped exons were detected at elevated frequencies in Rnps1F181I/F181I splenic CD8+ T cells and hematopoietic stem cells (HSCs), but NMD appeared normal. Strikingly, Tnf knockout rescued all hematopoietic cells to normal or near-normal levels in Rnps1F181I/F181I mice and dramatically reduced intron retention in Rnps1F181I/F181I CD8+ T cells and HSCs. Thus, RNPS1 is necessary for accurate splicing, without which disinhibited TNF signaling triggers hematopoietic cell death.

An Mtr4/ZFC3H1 complex facilitates turnover of unstable nuclear RNAs to prevent their cytoplasmic transport and global translational repression.

Many long noncoding RNAs (lncRNAs) are unstable and rapidly degraded in the nucleus by the nuclear exosome. An exosome adaptor complex called NEXT (nuclear exosome targeting) functions to facilitate turnover of some of these lncRNAs. Here we show that knockdown of one NEXT subunit, Mtr4, but neither of the other two subunits, resulted in accumulation of two types of lncRNAs: prematurely terminated RNAs (ptRNAs) and upstream antisense RNAs (uaRNAs). This suggested a NEXT-independent Mtr4 function, and, consistent with this, we isolated a distinct complex containing Mtr4 and the zinc finger protein ZFC3H1. Strikingly, knockdown of either protein not only increased pt/uaRNA levels but also led to their accumulation in the cytoplasm. Furthermore, all pt/uaRNAs examined associated with active ribosomes, but, paradoxically, this correlated with a global reduction in heavy polysomes and overall repression of translation. Our findings highlight a critical role for Mtr4/ZFC3H1 in nuclear surveillance of naturally unstable lncRNAs to prevent their accumulation, transport to the cytoplasm, and resultant disruption of protein synthesis.

Publisher Correction: ANKRD16 prevents neuron loss caused by an editing-defective tRNA synthetase.

In the Fig. 3b western blot of this Article, 'Myc-AlaRS' in row one should have been 'Myc-AAD Aars', 'AlaRS' in row two should have been 'Aars' and 'ANKRD16' in row four should have been 'Ankrd16'. In Fig. 4f, 'ANKRD16' and 'ANKRD16(3xR)' should have been 'Ankrd16' and 'Ankrd163xR; and in Fig. 3c the position of the molecular mass markers had shifted. These figures have been corrected online, and see Supplementary Information to the accompanying Amendment for the original figure.

ANKRD16 prevents neuron loss caused by an editing-defective tRNA synthetase.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars sti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.

A short ORF-encoded transcriptional regulator.

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

The proper connection between shelterin components is required for telomeric heterochromatin assembly.

Telomeric regions contain prominent sites of heterochromatin, which is associated with unique histone modification profiles such as the methylation of histone H3 at Lys9 (H3K9me). In fission yeast, the conserved telomeric shelterin complex recruits the histone H3K9 methyltransferase complex CLRC to establish subtelomeric heterochromatin. Although many shelterin mutations affect subtelomeric heterochromatin assembly, the mechanism remains elusive due to the diverse functions of shelterin. Through affinity purification, we found that shelterin directly associates with CLRC through the Ccq1 subunit. Surprisingly, mutations that disrupt interactions between shelterin subunits compromise subtelomeric heterochromatin without affecting CLRC interaction with shelterin component Pot1, located at chromosome ends. We further discovered that telomeric repeats are refractory to heterochromatin spreading and that artificial restoration of shelterin connections or increased heterochromatin spreading rescued heterochromatin defects in these shelterin mutants. Thus, subtelomeric heterochromatin assembly requires both the recruitment of CLRC by shelterin to chromosome ends and the proper connection of shelterin components, which allows CLRC to skip telomeric repeats to internal regions.

Cross-editing by a tRNA synthetase allows vertebrates to abundantly express mischargeable tRNA without causing mistranslation.

The accuracy in pairing tRNAs with correct amino acids by aminoacyl-tRNA synthetases (aaRSs) dictates the fidelity of translation. To ensure fidelity, multiple aaRSs developed editing functions that remove a wrong amino acid from tRNA before it reaches the ribosome. However, no specific mechanism within an aaRS is known to handle the scenario where a cognate amino acid is mischarged onto a wrong tRNA, as exemplified by AlaRS mischarging alanine to G4:U69-containing tRNAThr. Here, we report that the mischargeable G4:U69-containing tRNAThr are strictly conserved in vertebrates and are ubiquitously and abundantly expressed in mammalian cells and tissues. Although these tRNAs are efficiently mischarged, no corresponding Thr-to-Ala mistranslation is detectable. Mistranslation is prevented by a robust proofreading activity of ThrRS towards Ala-tRNAThr. Therefore, while wrong amino acids are corrected within an aaRS, a wrong tRNA is handled in trans by an aaRS cognate to the mischarged tRNA species. Interestingly, although Ala-tRNAThr mischarging is not known to occur in bacteria, Escherichia coli ThrRS also possesses robust cross-editing ability. We propose that the cross-editing activity of ThrRS is evolutionarily conserved and that this intrinsic activity allows G4:U69-containing tRNAThr to emerge and be preserved in vertebrates to have alternative functions without compromising translational fidelity.

CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3' processing.

AAUAAA is the most highly conserved motif in eukaryotic mRNA polyadenylation sites and, in mammals, is specifically recognized by the multisubunit CPSF (cleavage and polyadenylation specificity factor) complex. Despite its critical functions in mRNA 3' end formation, the molecular basis for CPSF-AAUAAA interaction remains poorly defined. The CPSF subunit CPSF160 has been implicated in AAUAAA recognition, but direct evidence has been lacking. Using in vitro and in vivo assays, we unexpectedly found that CPSF subunits CPSF30 and Wdr33 directly contact AAUAAA. Importantly, the CPSF30-RNA interaction is essential for mRNA 3' processing and is primarily mediated by its zinc fingers 2 and 3, which are specifically targeted by the influenza protein NS1A to suppress host mRNA 3' processing. Our data suggest that AAUAAA recognition in mammalian mRNA 3' processing is more complex than previously thought and involves multiple protein-RNA interactions.

A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation.

HIV-1 Tat stimulates transcription elongation by recruiting the P-TEFb (positive transcription elongation factor-b) (CycT1:CDK9) C-terminal domain (CTD) kinase to the HIV-1 promoter. Here we show that Tat transactivation also requires the Ssu72 CTD Ser5P (S5P)-specific phosphatase, which mediates transcription termination and intragenic looping at eukaryotic genes. Importantly, HIV-1 Tat interacts directly with Ssu72 and strongly stimulates its CTD phosphatase activity. We found that Ssu72 is essential for Tat:P-TEFb-mediated phosphorylation of the S5P-CTD in vitro. Interestingly, Ssu72 also stimulates nascent HIV-1 transcription in a phosphatase-dependent manner in vivo. Chromatin immunoprecipitation (ChIP) experiments reveal that Ssu72, like P-TEFb and AFF4, is recruited by Tat to the integrated HIV-1 proviral promoter in TNF-α signaling 2D10 T cells and leaves the elongation complex prior to the termination site. ChIP-seq (ChIP combined with deep sequencing) and GRO-seq (genome-wide nuclear run-on [GRO] combined with deep sequencing) analysis further reveals that Ssu72 predominantly colocalizes with S5P-RNAPII (RNA polymerase II) at promoters in human embryonic stem cells, with a minor peak in the terminator region. A few genes, like NANOG, also have high Ssu72 at the terminator. Ssu72 is not required for transcription at most cellular genes but has a modest effect on cotranscriptional termination. We conclude that Tat alters the cellular function of Ssu72 to stimulate viral gene expression and facilitate the early S5P-S2P transition at the integrated HIV-1 promoter.