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Type I interferon (IFN) drives pathology in systemic lupus erythematosus (SLE) and can be tracked via IFN-inducible transcripts in blood. Here, we examined whether measurement of circulating proteins, which enter the bloodstream from inflamed tissues, also offers insight into global IFN activity. Using a novel protocol we generated 1,132 aptamer-based protein measurements from anti-dsDNApos SLE blood samples and derived an IFN protein signature (IFNPS) that approximates the IFN 21-gene signature (IFNGS). Of 82 patients with SLE, IFNPS was elevated for 89% of IFNGS-high patients (49/55) and 26% of IFNGS-low patients (7/27). IFNGS-high/IFNPS-high patients exhibited activated NK, CD4, and CD8 T cells, while IFNPS-high only patients did not. IFNPS correlated with global disease activity in lymphopenic and non-lymphopenic patients and decreased following type I IFN neutralisation with anifrolumab in the SLE phase IIb study, MUSE. In summary, we developed a protein signature that reflects IFNGS and identifies a new subset of patients with SLE who have IFN activity.

Type I interferon (IFN) signaling drives pathology in systemic lupus erythematosus (SLE) and can be tracked via IFN-inducible transcripts present in whole blood as described by several IFN gene signatures. As SLE is a complex disease affecting diverse organ systems, we examined whether measurement of circulating proteins, which can infiltrate the bloodstream from afflicted tissues, might also offer insight into global IFN activity. The presence of anti-DNA autoantibodies in patient serum has prevented effective use of SOMAmers for the evaluation of circulating proteins in SLE. Here, we adapted protocols to mitigate for those autoantibodies and report high reproducibility and accuracy with 100% QC pass rate and improved correlation with previously validated multi-analyte platform results. Using SOMAmers together with the IFN 21-gene signature1 (IFNGS), we derived an IFN protein signature that can approximate the IFNGS score. In a cohort of 82 SLE patients and 48 healthy donors, the protein signature was found elevated above healthy donors for most of IFNGS-high patients (49/55, 89%) and also for a subgroup of IFNGS-low patients (7/27, 26%). The protein signature correlated with global disease activity (median SLEDAI score of 4 for the cohort) in both lymphopenic and non-lymphopenic patients. Significant associations with skin involvement, low complement, anti DNA auto-antibodies, and thrombocytopenia were also observed. In sum, our results suggest blood derived protein measurements may complement validated gene signatures to monitor IFN activity.

The Mauriceville and closely related Varkud plasmids are small circular DNAs (3.6 and 3.7 kb, respectively) found in the mitochondria of certain Neurospora spp. strains isolated from nature. The plasmids replicate via reverse transcription and appear to be primitive retroelements that may be related to the early ancestors of retroviruses. Recent studies have shown that the plasmid reverse transcriptase closely resembles certain viral RNA-dependent RNA polymerases in initiating (−) strand cDNA synthesis de novo (i.e., without a primer) at a tRNA-like structure at the 3′ end of the plasmid transcript. The plasmid reverse transcriptase can also use DNA or RNA primers and can carry out template-switching reactions that lead to the generation of suppressive mutant plasmids or the integration of the plasmids into mitochondrial DNA. The characteristics of the plasmids and their reverse transcription mechanism suggest an evolutionary connection between RNA and DNA replication and raise the possibility that the plasmids are related to the earliest DNA-based life forms that emerged at the time of transition from an RNA to a DNA world. Key words: DNA synthesis, evolution, retrovirus, reverse transcriptase, RNA virus.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery.

The high degree of similarity between the mouse and human genomes is demonstrated through analysis of the sequence of mouse chromosome 16 (Mmu 16), which was obtained as part of a whole-genome shotgun assembly of the mouse genome. The mouse genome is about 10% smaller than the human genome, owing to a lower repetitive DNA content. Comparison of the structure and protein-coding potential of Mmu 16 with that of the homologous segments of the human genome identifies regions of conserved synteny with human chromosomes (Hsa) 3, 8, 12, 16, 21, and 22. Gene content and order are highly conserved between Mmu 16 and the syntenic blocks of the human genome. Of the 731 predicted genes on Mmu 16, 509 align with orthologs on the corresponding portions of the human genome, 44 are likely paralogous to these genes, and 164 genes have homologs elsewhere in the human genome; there are 14 genes for which we could find no human counterpart.

The Mauriceville retroplasmid of Neurospora mitochondria encodes a novel reverse transcriptase that initiates cDNA synthesis de novo (i.e., without a primer) at the 3' CCA of the plasmid transcript's 3' tRNA-like structure (H. Wang and A. M. Lambowitz, Cell 75:1071-1081, 1993). Here, we show that the plasmid reverse transcriptase also initiates cDNA synthesis de novo at the 3' end of tRNAs, leading to synthesis of a full-length cDNA copy of the tRNA. The use of tRNA templates in vivo was suggested previously by the structure of suppressive mutant plasmids that have incorporated mitochondrial tRNA sequences (R. A. Akins, R. L. Kelley, and A. M. Lambowitz, Cell 47:505-516, 1986). The in vitro experiments show that efficient de novo initiation on tRNA templates requires an unpaired 3' CCA and occurs predominantly opposite position C-2 of the 3' CCA sequence, the same position as in the plasmid transcript. In other reactions, the plasmid reverse transcriptase synthesizes cDNA dimers by template switching between two tRNA templates and initiates at an internal position in a tRNA by using the 3' end of the tRNA as a primer. Finally, we show that template switching between the tRNA and the plasmid transcript in vitro gives rise to hybrid cDNAs of the type predicted to be intermediates in the generation of the suppressive mutant plasmids. The ability of the plasmid reverse transcriptase to initiate at the 3' end of tRNAs presumably reflects the recognition of structural features similar to those of the 3' tRNA-like structure of the plasmid transcript. The recognition of tRNAs or tRNA-like structures as templates for cDNA synthesis may be characteristic of primitive reverse transcriptases that evolved from RNA-dependent RNA polymerases.