Biology subjects

Pfleiderer, M. M.

Publications and source records attributed to Pfleiderer, M. M..

4 recordsLinked to original sources

bioRxiv2025

Ashwin and FAM98 paralogs define nuclear and cytoplasmic RNA ligase complexes for tRNA biogenesis and the unfolded protein response

The tRNA ligase complex (tRNA-LC) seals tRNA exon halves in the nucleus after the removal of a single intron, and joins XBP1-mRNA exons in the cytoplasm as part of the unfolded protein response (UPR). This dual function requires simultaneous nuclear and cytoplasmic localization. Here, we reveal that Ashwin (ASW), the vertebrate-specific subunit of the tRNA-LC, serves as its nuclear import factor. ASW displays a dual nuclear localisation signal (NLS) which, upon disruption, leads to the retention of the tRNA-LC in the cytoplasm with a consequent impairment of pre-tRNA splicing and accumulation of 5 tRNA fragments. We also show that the tRNA-LC exists in three forms depending on which FAM98 paralog is chosen, either FAM98A, FAM98B or FAM98C. We find that ASW interacts exclusively with the FAM98B-containing complex, allowing its nuclear localization for tRNA biogenesis. Attaching an NLS to RTCB, the catalytic and indispensable subunit, rescues pre-tRNA splicing in cells depleted of ASW. We envision that vertebrates evolved ASW to localize a sub-population of tRNA-LC to the nucleus, while using FAM98 paralogs to retain a fraction of RTCB in the cytoplasm for XBP1-mRNA splicing during UPR.

cell biology↗

bioRxiv2025

Structural framework for the assembly of the human tRNA ligase complex

In human cells, a subset of tRNA-encoding genes contain introns. These are removed by a non-canonical splicing pathway in which the tRNA splicing endonuclease complex catalyzes intron excision and the resulting exons are subsequently ligated by the tRNA-ligase complex (tRNA-LC). Although recent studies have provided insights into the process of intron removal, the molecular mechanisms underpinning tRNA ligation by tRNA-LC remain elusive. The tRNA-LC is a hetero-pentameric protein assembly consisting of Ashwin, CGI-99, FAM98B, the DEAD-box helicase DDX1 and the catalytic subunit RTCB/HSPC117. Using cryo-EM, we have determined an atomic-resolution reconstruction of human tRNA-LC. We find that CGI-99, DDX1 and FAM98B form an alpha-helical bundle that contacts RTCB via an interface located on the opposite side from the location of the ligase active site and tethers DDX1 to the tRNA-LC via its C-terminal helix. FAM98B and CGI-99 extensively interact in an intricately co-folded heterodimer that clamps Ashwin in a pincer-like structure. Interaction analysis using structure-based mutants of tRNA-LC subunits supports the overall architecture of the complex. Finally, we show that the paralogous proteins FAM98A and FAM98C underpin the assembly of compositionally distinct RTCB-containing complexes that lack Ashwin and may have distinct cellular functions. Together, our results provide new insights into the assembly and mechanism of the tRNA ligase complex, shedding light on its functions in tRNA biogenesis and beyond.

biochemistry↗

bioRxiv2024

Structural basis of the Integrator complex assembly and association with transcription factors

Integrator is a multi-subunit protein complex responsible for premature transcription termination of coding and non-coding RNAs in Metazoans. This is achieved via Integrators two enzymatic activities, RNA endonuclease and protein phosphatase, acting on the promoter-proximally paused RNA Polymerase II (RNAPII). Yet, it remains unclear how Integrator assembly and recruitment are regulated and what are the functions of many of its core subunits. Here we report two cryo-EM reconstructions of large Integrator sub-complexes: INTS10/13/14/15 (Arm module) and INTS5/8/10/15, which allowed integrative modelling of the fully-assembled Integrator bound to the RNAPII paused elongating complex (PEC). INTS13/14 are positioned near the DNA upstream from the transcription pause site, suggesting a potential role in the chromatin context. An in silico protein interaction screen of over 1500 transcription factors (TFs), identified Zinc Finger Protein 655 (ZNF655) as a direct interacting partner of INTS13 that associates with a fully assembled, 17-subunit Integrator complex. We propose a model wherein the Arm module acts as a platform for the recruitment of TFs that could modulate the stability of the Integrators association at specific loci and modulate transcription attenuation of the target genes.

biochemistry↗

bioRxiv2023

Mechanistic basis for oxidative stress protection of the human tRNA ligase complex by the oxidoreductase PYROXD1

RTCB is the catalytic subunit of the metazoan tRNA ligase complex (tRNA-LC) that plays essential roles in tRNA biogenesis and unfolded protein response. The catalytic center of RTCB contains a conserved cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of mammalian tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here we report a cryo-EM structure of human RTCB in complex with PYROXD1, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its C-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment and PYROXD1, while PYROXD1 reoxidation enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of tRNA-LC under aerobic conditions.

biochemistry↗