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A POLR3B-variant reveals a Pol III transcriptome response dependent on La protein/SSB.

ABSTRACT: RNA polymerase III (Pol III, POLR3) synthesizes tRNAs and other small non-coding RNAs. Human POLR3 pathogenic variants cause a range of developmental disorders, recapitulated in part by mouse models, yet some aspects of POLR3 deficiency have not been explored. We characterized a human POLR3B:c.1625A>G;p.(Asn542Ser) disease variant that was found to cause mis-splicing of POLR3B. Genome-edited POLR3B^1625A>G HEK293 cells acquired the mis-splicing with decreases in multiple POLR3 subunits and TFIIIB, although display auto-upregulation of the Pol III termination-reinitiation subunit POLR3E. La protein was increased relative to its abundant pre-tRNA ligands which bind via their U(n)U-3'-termini. Assays for cellular transcription revealed greater deficiencies for tRNA genes bearing terminators comprised of 4Ts than of ≥5Ts. La-knockdown decreased Pol III ncRNA expression unlinked to RNA stability. Consistent with these effects, small-RNAseq showed that POLR3B^1625A>G and patient fibroblasts express more tRNA fragments (tRFs) derived from pre-tRNA 3'-trailers (tRF-1) than from mature-tRFs, and higher levels of multiple miRNAs, relative to control cells. The data indicate that decreased levels of Pol III transcripts can lead to functional excess of La protein which reshapes small ncRNA profiles revealing new depth in the Pol III system. Finally, patient cell RNA analysis uncovered a strategy for tRF-1/tRF-3 as POLR3-deficiency biomarkers.

SUBMITTER: Mattijssen S

PROVIDER: S-EPMC10896340 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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A POLR3B-variant reveals a Pol III transcriptome response dependent on La protein/SSB.

Mattijssen Sandy S Kerkhofs Kyra K Stephen Joshi J Yang Acong A Han Chen G CG Tadafumi Yokoyama Y Iben James R JR Mishra Saurabh S Sakhawala Rima M RM Ranjan Amitabh A Gowda Mamatha M Gahl William A WA Gu Shuo S Malicdan May C MC Maraia Richard J RJ

bioRxiv : the preprint server for biology 20240205

RNA polymerase III (Pol III, POLR3) synthesizes tRNAs and other small non-coding RNAs. Human POLR3 pathogenic variants cause a range of developmental disorders, recapitulated in part by mouse models, yet some aspects of POLR3 deficiency have not been explored. We characterized a human POLR3B:c.1625A>G;p.(Asn542Ser) disease variant that was found to cause mis-splicing of POLR3B. Genome-edited POLR3B1625A>G HEK293 cells acquired the mis-splicing with d ...[more]

PMID: 38410490

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Project description:BACKGROUND: Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. MATERIALS AND METHODS: Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. RESULTS: All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. CONCLUSIONS: Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a common anti-La/SSB idiotype. These antibodies can be detected using complementary peptides of La/SSB epitopes. The antiidiotypic antibodies mask the anti-La/SSB response. Hidden anti-La/SSB antibodies can be released and detected using complementary epitope analogs.

Project description:Translesion synthesis (TLS) by specialized DNA polymerases (Pols) is an evolutionarily conserved mechanism for tolerating replication-blocking DNA lesions. Using the Escherichia coli dinB-encoded Pol IV as a model to understand how TLS is coordinated with the actions of the high-fidelity Pol III replicase, we previously described a novel Pol IV mutant containing a threonine 120-to-proline mutation (Pol IV-T120P) that failed to exchange places with Pol III at the replication fork in vitro as part of a Pol III-Pol IV switch. This in vitro defect correlated with the inability of Pol IV-T120P to support TLS in vivo, suggesting Pol IV gains access to the DNA, at least in part, via a Pol III-Pol IV switch. Interaction of Pol IV with the β sliding clamp and the single-stranded DNA binding protein (SSB) significantly stimulates Pol IV replication and facilitates its access to the DNA. In this work, we demonstrate that Pol IV interacts physically with Pol III. We further show that Pol IV-T120P interacts normally with the β clamp, but is impaired in interactions with the α catalytic and εθ proofreading subunits of Pol III, as well as SSB. Taken together with published work, these results provide strong support for the model in which Pol IV-Pol III and Pol IV-SSB interactions help to regulate the access of Pol IV to the DNA. Finally, we describe several additional E. coli Pol-Pol interactions, suggesting Pol-Pol interactions play fundamental roles in coordinating bacterial DNA replication, DNA repair, and TLS. IMPORTANCE Specialized DNA polymerases (Pols) capable of catalyzing translesion synthesis (TLS) generate mutations that contribute to bacterial virulence, pathoadaptation, and antimicrobial resistance. One mechanism by which the bacterial TLS Pol IV gains access to the DNA to generate mutations is by exchanging places with the bacterial Pol III replicase via a Pol III-Pol IV switch. Here, we describe multiple Pol III-Pol IV interactions and discuss evidence that these interactions are required for the Pol III-Pol IV switch. Furthermore, we describe several additional E. coli Pol-Pol interactions that may play fundamental roles in managing the actions of the different bacterial Pols in DNA replication, DNA repair, and TLS.

Project description:Systemic lupus erythematosus (SLE) is characterized by the production of grouped sets of autoantibodies targeting mainly the U1 ribonucleoprotein (RNP) and/or Ro/La RNP particles. Intraparticle diversification of the autoimmune response is believed to occur via epitope spreading. So far, it is not known how the autoimmune response "jumps" from one particle to another. To the extent that the majority of nuclear autoantigens in SLE are RNA binding proteins and major epitopes were previously mapped within their RRM (RNA recognition motifs), conserved sequences within RRM could be involved in the intermolecular and inter-particle diversification process of the autoimmune response. We investigated the potential of RRM of the La/SSB autoantigen to induce antibodies that cross-recognize components of the U1-RNP particle and therefore its capacity to produce interparticle epitope spreading. We immunized New Zealand white rabbits with a peptide corresponding to the epitope 145-164 of La/SSB (belonging to the RRM of La/SSB), attached in four copies on a scaffold carrier. Sera were drawn from 20 sera of patients with SLE and anti-U1-RNP antibodies and 26 sera of primary Sjögren syndrome patients with anti-La/SSB antibodies. All sera were evaluated for reactivity against the major epitope of La/SSB (pep349-364), the RNP antigen and the RRM-related epitope of La/SSB (pep145-164). Specific antibodies against pep145-164 were purified with immunoaffinity columns from selected sera. After the immunization of the animals with pep145-164, a specific IgG antibody response was detected, directed against the La/SSB autoantigen (wks 3-7), the immunizing peptide (wks 3-27), and the RNP autoantigen (wks 7-20). This response gradually decreased to low levels between postimmunization wks 27-42. Purified antibodies against pep145-164 recognized La/SSB and a 70-kD autoantigen in Western blot and exhibited significant reactivity in anti-U1-RNP ELISA. Depletion of anti-pep145-164 antibodies eliminated anti-U1-RNP reactivity from immunized rabbit sera but not from human sera. In addition, pep145-164 was recognized to a greater extent by autoimmune sera with anti-RNP reactivity compared with anti-La/SSB-positive sera, in contrast to pep349-364 of La/SSB, which was recognized almost exclusively by sera with anti-La/SSB reactivity. These data suggest that the RRM region of La/SSB can trigger interparticle B-cell diversification to U1-RNP-70 autoantigen via molecular mimicry. Identification of key sequences that trigger and perpetuate the autoimmune process is particularly important for understanding pathogenetic mechanisms in autoimmunity.

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A POLR3B-variant reveals a Pol III transcriptome response dependent on La protein/SSB.

Publications

A <i>POLR3B</i>-variant reveals a Pol III transcriptome response dependent on La protein/SSB.

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