Clinical outcome upon infection with SARS-CoV-2 ranges from silent infection to lethal COVID-19. We have found an enrichment in rare variants predicted to be loss-of-function (LOF) at the 13 human loci known to govern TLR3- and IRF7-dependent type I interferon (IFN) immunity to influenza virus, in 659 patients with life-threatening COVID-19 pneumonia, relative to 534 subjects with asymptomatic or benign infection. By testing these and other rare variants at these 13 loci, we experimentally define LOF variants in 23 patients (3.5%), aged 17 to 77 years, underlying autosomal recessive or dominant deficiencies. We show that human fibroblasts with mutations affecting this pathway are vulnerable to SARS-CoV-2. Inborn errors of TLR3- and IRF7-dependent type I IFN immunity can underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection.
We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation-contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation-contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.T he expression of more than 95% of human genes is affected by alternative pre-mRNA splicing (AS) (1, 2). Differentially spliced isoforms play distinct roles in a temporally and spatially specific manner (3), and mutations that lead to aberrant splicing are the cause of many human genetic diseases (4). RNA-binding proteins (RBPs) play a central role in the regulation of alternative splicing during development and disease. They function primarily by positively or negatively regulating splice-site recognition by the spliceosome (1). Many RBPs are expressed in specific tissues, and AS is regulated by the combinatorial activities of these factors on specific pre-mRNAs through their interactions with distinct regulatory sequences in premRNA that function as splicing enhancers or silencers (5).The developing heart is one of the best studied systems where splicing changes occur during normal development, and mutations affecting specific splicing outcomes contribute to cardiomyopathy (6, 7). Although these mutations can either disrupt splicing elements or affect the expression of specific splicing factors, the latter mechanism is clearly responsible for the distinct splicing profiles at different developmental stages. For example, the dynamics of alternative splicing during postnatal heart development correlate with expression changes of many RBPs, including CUG-BP, Elavlike family member 1 (CELF1), Muscleblind-like 1 (MBNL1), and FOX proteins (8). Detailed biochemical studies have elucidated the mechanisms by which these splicing factors regulate splicing in a position-and context-dependent manner (9, 10). The function of other RBPs during heart development has also been studied. For example, two of the muscle-specific splicing factors, RBM20 and RBM24, play distinct roles in splicing regulation. RBM20 mainly acts as a splicing repressor, as its absence leads to multiple exon inclusion events in the heart. For example, the Titin gene is one of...
R2 retrotransposable elements exclusively insert into a conserved region of the tandemly organized 28S rRNA genes. Despite inactivating a subset of these genes, R2 elements have persisted in the ribosomal DNA (rDNA) loci of insects for hundreds of millions of years. Controlling R2 proliferation was addressed in this study using lines of Drosophila simulans previously shown to have either active or inactive R2 retrotransposition. Lines with active retrotransposition were shown to have high R2 transcript levels, which nuclear run-on transcription experiments revealed were due to increased transcription of R2-inserted genes. Crosses between R2 active and inactive lines indicated that an important component of this transcriptional control is linked to or near the rDNA locus, with the R2 transcription level of the inactive parent being dominant. Pulsed-field gel analysis suggested that the R2 active and inactive states were determined by R2 distribution within the locus. Molecular and cytological analyses further suggested that the entire rDNA locus from the active line can be silenced in favor of the locus from the inactive line. This silencing of entire rDNA loci represents an example of the large-scale epigenetic control of transposable elements and shares features with the nucleolar dominance frequently seen in interspecies hybrids.Eukaryotic genomes have evolved elaborate surveillance and regulatory mechanisms to control the spread of transposable elements (38). The success of a transposable element is thus dependent upon its ability to elude these cellular controls. One ingenious approach used by transposable elements is to target locations within the genome that cannot be completely silenced. The most successful known examples of this approach are the numerous mobile elements that insert specifically into the rRNA genes of animals (10). Eukaryotic genomes encode hundreds to thousands of rRNA genes organized in tandem arrays within one or more chromosomal loci. Transcription of the rRNA gene arrays, also termed nucleolar organizer regions (NORs), is tightly coupled to the growth status of the cell. Significant progress has been made in understanding the transcription of the DNA encoding the rRNA genes (ribosomal DNA [rDNA]) as well as the many subsequent steps involved in ribosome biogenesis (14, 16). The level of regulatory complexity added by the presence of transposable elements is largely unknown.The nonlong terminal repeat (non-LTR) retrotransposable elements, R1 and R2, insert into a conserved central region of the 28S gene (Fig. 1A). R1 and R2 are present in most lineages of arthropods (2), and R2 elements have been found in a number of other divergent animal groups (20, 21). Phylogenetic analyses suggest that R2 elements have been a stable component of genomes throughout the evolution of arthropods (2, 27) and possibly since the origin of multicellular animals (20, 21). As such, they represent the longest known stable relationship of a mobile element and its host. During their long history, R2 elements ...
Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5 end of the R2 element. In the absence of this 5 RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3 untranslated region of the R2 transcript is present. In the presence of the 320-nt 5 RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5 RNA is removed from this subunit. We postulate that the removal of the 5 RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first-and second-strand DNA cleavage in the R2 retrotransposition reaction.endonuclease ͉ retrotransposition ͉ reverse transcription ͉ RNA-protein interactions N on-LTR retrotransposons, also referred to as long interspersed nuclear elements (LINEs), are abundant insertions in many eukaryotic genomes. For example, there are Ͼ800,000 copies of these elements in the human genome, representing 17% of our DNA (1). Whereas retrotransposition assays in tissue culture cells have been developed to study non-LTR retrotransposition, many questions concerning the mechanism of their integration remain unanswered (2-5).R2 is a non-LTR retrotransposable element with rigid sequence specificity for a target site in the 28S rRNA genes of arthropods, platyhelminths, tunicates, and vertebrates (6, 7). The sequence specificity of R2 integration has enabled detailed biochemical studies of its retrotransposition reaction (Fig. 1A). We have previously shown that one R2 protein subunit of a probable dimer binds a 30-bp DNA segment upstream of the insertion site and cleaves the first strand (bottom strand, Fig. 1 A) of the target DNA (8, 9). If RNA corresponding to the 3Ј UTR of the R2 element is present, then this subunit primes reverse transcription of the R2 RNA transcript from the free 3Ј end released by the cleavage. This process is referred to as targetprimed reverse transcription (TPRT) (10). After reverse transcription, the second (top) DNA strand is cleaved by the second protein subunit, which binds a different DNA sequence downstream of the insertion site (9). We have postulated that this second R2 subunit is responsible for the synthesis of the second DNA strand and thereby completes the retrotransposition reaction (9).One ...
About half of the rRNA gene units (rDNA units) of Drosophila melanogaster are inserted by the retrotransposable elements R1 and R2. Because transcripts to R1 and R2 were difficult to detect on blots and electron microscopic observations of rRNA synthesis suggested that only uninserted rDNA units were transcribed, it has long been postulated that inserted rDNA units are in a repressed (inactive) chromatin structure. Studies described here suggest that inserted and uninserted units are equally accessible to DNase I and micrococcal nuclease and contain similar levels of histone H3 and H4 acetylation and H3K9 methylation. These studies have low sensitivity, because psoralen cross-linking suggested few (estimated <10%) of the rDNA units of any type are transcriptionally active. Nuclear run-on experiments revealed that R1-inserted and R2-inserted units are activated for transcription at about 1/5 and 1/10, respectively, the rate of uninserted units. Most transcription complexes of the inserted units terminate within the elements, thus explaining why previous molecular and electron microscopic methods indicated inserted units are seldom transcribed. The accumulating data suggest that all units within small regions of the rDNA loci are activated for transcription, with most control over R1 and R2 activity involving steps downstream of transcription initiation.Initial cloning of the repeated rRNA genes (rDNA units) of Drosophila melanogaster revealed large insertions within the 28S rRNA gene ( Fig. 1) (28,63). These sequences, originally termed type I and type II insertions, were eventually identified as two distinct lineages of site-specific non-long-terminal-repeat retrotransposons and renamed R1 and R2 (33). Typically half (range, 32 to 77%) of the rDNA units in different geographical strains of D. melanogaster are inserted by R1 or R2 (34). Full-length R1 and R2 insertions are 5.3 kb and 3.6 kb, respectively, but many insertions are truncated to variable extents at their 5Ј end. All elements are inserted in the same transcriptional orientation as the 28S gene. The remarkable specificity of each element is dependent upon the combined action of a specific endonuclease that cleaves the target site and a reverse transcriptase that uses this cleavage to prime reverse transcription (2,12,46).The sequences of inserted and uninserted rDNA units are identical (38, 45), a reflection of the frequent retrotransposition of R1 or R2 elements into the rDNA units and their elimination by recombination (4,34,53,54). In spite of this rapid turnover, phylogenetic analyses have revealed that these elements have been vertically maintained in insect lineages since the origin of arthropods (6,23,48). More recent studies have revealed R2 or related non-long-terminal-repeat retrotransposons inserted near the R1 and R2 sites in the rRNA genes of nematodes, platyhelminthes, tunicates, and vertebrates (7,8,21,39).Previous studies of inserted rDNA units have suggested that both the insertions and the rDNA units they inhabit are not transcribed. ...
Highlights d TBK1 kinase activity regulates disease progression in an ALS SOD1 mouse model d Loss of TBK1 in motor neurons increases SOD1 aggregation and accelerates disease onset d Loss of TBK1 activity in all cell types accelerates disease onset but extends survival d Loss of TBK1 activity in all cell types reduces the IFN response in microglia
Exonic DNA sequence variants in the Tbk1 gene associate with both sporadic and familial amyotrophic lateral sclerosis (ALS). Here, we examine functional defects in 25 missense TBK1 mutations, focusing on kinase activity and protein–protein interactions. We identified kinase domain (KD) mutations that abolish kinase activity or display substrate-specific defects in specific pathways, such as innate immunity and autophagy. By contrast, mutations in the scaffold dimerization domain (SDD) of TBK1 can cause the loss of kinase activity due to structural disruption, despite an intact KD. Familial ALS mutations in ubiquitin-like domain (ULD) or SDD display defects in dimerization; however, a subset retains kinase activity. These observations indicate that TBK1 dimerization is not required for kinase activation. Rather, dimerization seems to increase protein stability and enables efficient kinase–substrate interactions. Our study revealed many aspects of TBK1 activities affected by ALS mutations, highlighting the complexity of disease pathogenicity and providing insights into TBK1 activation mechanism.
During pregnancy, fetal extravillous trophoblasts (EVT) play a key role in the regulation of maternal T cell and NK cell responses. EVT display a unique combination of human leukocyte antigens (HLA); EVT do not express HLA-A and HLA-B, but do express HLA-C, HLA-E, and HLA-G. The mechanisms establishing this unique HLA expression pattern have not been fully elucidated. The major histocompatibility complex (MHC) class I and class II transcriptional activators NLRC5 and CIITA are expressed neither by EVT nor by the EVT model cell line JEG3, which has an MHC expression pattern identical to that of EVT. Therefore, other MHC regulators must be present to control HLA-C, HLA-E, and HLA-G expression in these cells. CIITA and NLRC5 are both members of the nucleotide-binding domain, leucine-rich repeat (NLR) family of proteins. Another member of this family, NLRP2, is highly expressed by EVT and JEG3, but not in maternal decidual stromal cells. In this study, transcription activator-like effector nuclease technology was used to delete NLRP2 in JEG3. Furthermore, lentiviral delivery of shRNA was used to knockdown NLRP2 in JEG3 and primary EVT. Upon NLRP2 deletion, Tumor Necrosis Factor-α (TNFα)-induced phosphorylation of NF-KB p65 increased in JEG3 and EVT, and more surprisingly a significant increase in constitutive HLA-C expression was observed in JEG3. These data suggest a broader role for NLR family members in the regulation of MHC expression during inflammation, thus forming a bridge between innate and adaptive immune responses. As suppressor of proinflammatory responses, NLRP2 may contribute to preventing unwanted antifetal responses.
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