The Alazami Syndrome-Associated Protein LARP7 Guides U6 Small Nuclear RNA Modification and Contributes to Splicing Robustness.

The La-related protein 7 (LARP7) forms a complex with the nuclear 7SK RNA to regulate RNA polymerase II transcription. It has been implicated in cancer and the Alazami syndrome, a severe developmental disorder. Here, we report a so far unknown role of this protein in RNA modification. We show that LARP7 physically connects the spliceosomal U6 small nuclear RNA (snRNA) with a distinct subset of box C/D small nucleolar RNAs (snoRNAs) guiding U6 2'-O-methylation. Consistently, these modifications are severely compromised in the absence of LARP7. Although general splicing remains largely unaffected, transcriptome-wide analysis revealed perturbations in alternative splicing in LARP7-depleted cells. Importantly, we identified defects in 2'-O-methylation of the U6 snRNA in Alazami syndrome siblings carrying a LARP7 mutation. Our data identify LARP7 as a bridging factor for snoRNA-guided modification of the U6 snRNA and suggest that alterations in splicing fidelity contribute to the etiology of the Alazami syndrome.

LARP7-Mediated U6 snRNA Modification Ensures Splicing Fidelity and Spermatogenesis in Mice.

U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2'-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2'-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2'-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2'-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2'-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.

Seed sequence polymorphism rs2168518 and allele-specific target gene regulation of hsa-miR-4513.

MicroRNAs (miRNAs) are small post-transcriptional regulators that offer promising targets for treating complex diseases. To this end, hsa-miR-4513 is an excellent candidate as this gene harbors within its conserved heptametrical seed sequence a frequent polymorphism (rs2168518), which has previously been associated with several complex phenotypes. So far, little is known about the biological mechanism(s) underlying these associations. In an initial step, we now aimed to identify allele-specific target genes of hsa-miR-4513. We performed RNA sequencing in a miRNA overexpression model in human umbilical vein endothelial cells transfected with separated hsa-miR-4513 alleles at rs2168518, namely hsa-miR-4513-G and hsa-miR-4513-A. Genes specifically regulated by the rs2168518 alleles were independently verified by quantitative reverse transcription PCR (qRT-PCR), western blot analysis and allele-specific miRNA binding via a luciferase reporter assay. By a text-based search publicly available databases such as Online Mendelian Inheritance in Man and Mouse Genome Informatics were utilized to link target genes of hsa-miR-4513 to previously described phenotypes. Overall, we identified 23 allele-specific hsa-miR-4513 target genes and replicated 19 of those independently via qRT-PCR. Western blot analysis and luciferase reporter assays conducted for an exemplary subsample further confirmed the allele-specific regulation of these genes by hsa-miR-4513. Remarkably, multiple allele-specific target genes identified are linked via text retrieval to several phenotypes previously reported to be associated with hsa-miR-4513. These genes offer promising candidates for ongoing research on the functional pathobiological impact of hsa-miR-4513 and its seed polymorphism rs2168518. This could give rise to therapeutic applications targeting this miRNA.

The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription.

Inhibition of host cell apoptosis is crucial for survival and replication of several intracellular bacterial pathogens. To interfere with apoptotic pathways, some pathogens use specialized secretion systems to inject bacterial effector proteins into the host cell cytosol. One of these pathogens is the obligate intracellular bacterium Coxiella burnetii, the etiological agent of the zoonotic disease Q fever. In this study, we analyzed the molecular activity of the anti-apoptotic T4SS effector protein AnkG (CBU0781) to understand how C. burnetii manipulates host cell viability. We demonstrate by co- and RNA-immunoprecipitation that AnkG binds to the host cell DExD box RNA helicase 21 (DDX21) as well as to the host cell 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, an important regulator of the positive transcription elongation factor b (P-TEFb). The co-immunoprecipitation of AnkG with DDX21 is probably mediated by salt bridges and is independent of AnkG-7SK snRNP binding, and vice versa. It is known that DDX21 facilitates the release of P-TEFb from the 7SK snRNP complex. Consistent with the documented function of released P-TEFb in RNA Pol II pause release, RNA sequencing experiments confirmed AnkG-mediated transcriptional reprogramming and showed that expression of genes involved in apoptosis, trafficking, and transcription are influenced by AnkG. Importantly, DDX21 and P-TEFb are both essential for AnkG-mediated inhibition of host cell apoptosis, emphasizing the significance of the interaction of AnkG with both, the DDX21 protein and the 7SK RNA. In line with a critical function of AnkG in pathogenesis, the AnkG deletion C. burnetii strain was severely affected in its ability to inhibit host cell apoptosis and to generate a replicative C. burnetii-containing vacuole. In conclusion, the interference with the activity of regulatory host cell RNAs mediated by a bacterial effector protein represent a novel mechanism through which C. burnetii modulates host cell transcription, thereby enhancing permissiveness to bacterial infection.

The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway.

The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading.

Stabilize and connect: the role of LARP7 in nuclear non-coding RNA metabolism.

La and La-related proteins (LARPs) are characterized by a common RNA interaction platform termed the La module. This structural hallmark allows LARPs to pervade various aspects of RNA biology. The metazoan LARP7 protein binds to the 7SK RNA as part of a 7SK small nuclear ribonucleoprotein (7SK snRNP), which inhibits the transcriptional activity of RNA polymerase II (Pol II). Additionally, recent findings revealed unanticipated roles of LARP7 in the assembly of other RNPs, as well as in the modification, processing and cellular transport of RNA molecules. Reduced levels of functional LARP7 have been linked to cancer and Alazami syndrome, two seemingly unrelated human diseases characterized either by hyperproliferation or growth retardation. Here, we review the intricate regulatory networks centered on LARP7 and assess how malfunction of these networks may relate to the etiology of LARP7-linked diseases.

An argonaute protein directs nuclear Xrn2 function.

Argonaute proteins are the mediators of small RNA-guided gene silencing pathways. In this issue, Couvillion and coworkers (Couvillion et al., 2012) found an unexpected function for a Tetrahymena Argonaute protein: It forms a complex with tRNA fragments and is required for nuclear Xrn2 localization and function.

From tRNA to miRNA: RNA-folding contributes to correct entry into noncoding RNA pathways.

Although RNA are synthesized as single-stranded molecules, most of them are characterized by extensive secondary structures. Two RNA that require distinct folding for biogenesis and function are miRNA and tRNA. While miRNA are processed from hairpin-containing precursors, tRNA are folded into characteristic L-shaped structures. In addition, tRNA and their precursors are a rich source of RNA fragments. Recent findings suggest that their production might be determined by structural characteristics of the tRNA substrates. Importantly, correct folding of pre-tRNA is assisted by the Lupus autoantigen La, an RNA chaperone. In this context, La interacts with pre-tRNA to prevent alternative foldings leading to mis-channeling into the miRNA pathway. Thus, RNA chaperones also function as gatekeepers for correct RNA pathway selection.

Epstein-Barr Virus EBER Transcripts Affect miRNA-Mediated Regulation of Specific Targets and Are Processed to Small RNA Species.

The oncogenic Epstein-Barr virus (EBV) expresses 44 mature microRNAs and two non-coding EBER RNAs of 167 (EBER1) and 172 (EBER2) nt length. MiRNA profiling of NK/T cell lines and primary cells and Northern blotting of EBV-infected cell lines and primary tumors revealed processing of EBER1 to short 5'-derived RNAs of approximately 23, 52 and 70 nt (EBER123, EBER152, and EBER170) and of EBER2 to 3' fragments. The biogenesis of these species is independent of Dicer, and EBER123 does not act like a miRNA OPEN ACCESS Non-Coding RNA 2015, 1 171 to target its complementary sequence. EBER1, EBER2 and EBER123 were bound by the lupus antigen (La), a nuclear and cytoplasmic protein that facilitates RNAi. Consistent with this, the EBERs affect regulation of interleukin 1alpha (IL1α) and RAC1 reporters harboring miR target sequences, targets of miR-142-3p. However, the EBERs have no effect upon another target of miR-142-3p, ADCY9, nor on TOMM22, a target of ebv-miR-BART16, indicative of selective modulation of gene expression by the EBERs.

A circulating microrna profile is associated with late-stage neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of severe vision impairment in Western populations over 55 years. A growing number of gene variants have been identified which are strongly associated with an altered risk to develop AMD. Nevertheless, gene-based biomarkers which could be dysregulated at defined stages of AMD may point toward key processes in disease mechanism and thus may support efforts to design novel treatment regimens for this blinding disorder. Circulating microRNAs (cmiRNAs) which are carried by nanosized exosomes or microvesicles in blood plasma or serum, have been recognized as valuable indicators for various age-related diseases. We therefore aimed to elucidate the role of cmiRNAs in AMD by genome-wide miRNA expression profiling and replication analyses in 147 controls and 129 neovascular AMD patients. We identified three microRNAs differentially secreted in neovascular (NV) AMD (hsa-mir-301-3p, pcorrected = 5.6*10-5, hsa-mir-361-5p, pcorrected = 8.0*10-4 and hsa-mir-424-5p, pcorrected = 9.6*10-3). A combined profile of the three miRNAs revealed an area under the curve (AUC) value of 0.727 and was highly associated with NV AMD (p = 1.2*10-8). To evaluate subtype-specificity, an additional 59 AMD cases with pure unilateral or bilateral geographic atrophy (GA) were analyzed for microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p. While we found no significant differences between GA AMD and controls neither individually nor for a combined microRNAs profile, hsa-mir-424-5p levels remained significantly higher in GA AMD when compared to NV (pcorrected<0.005). Pathway enrichment analysis on genes predicted to be regulated by microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p, suggests canonical TGFß, mTOR and related pathways to be involved in NV AMD. In addition, knockdown of hsa-mir-361-5p resulted in increased neovascularization in an in vitro angiogenesis assay.