A unified mechanism for intron and exon definition and wait on-splicing

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Abstract

The molecular mechanisms of exon definition and wait on-splicing are foremost unanswered questions in pre-mRNA splicing. Right here we file cryo-electron microscopy structures of the yeast spliceosomal E complex assembled on introns, offering a peep of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex structure means that the same spliceosome can assemble across an exon, and that it both remodels to span an intron for canonical linear splicing (on the total on short exons) or catalyses wait on-splicing to generate spherical RNA (on lengthy exons). The model is supported by our experiments, which display that an E complex assembled on the center exon of yeast EFM5 or HMRA1 can also even be chased into spherical RNA when the exon is sufficiently lengthy. This straightforward model unifies intron definition, exon definition, and wait on-splicing thru the same spliceosome in all eukaryotes and might per chance per chance well inspire experiments in many other systems to achieve the mechanism and regulation of these processes.

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Records availability

The coordinate recordsdata were deposited in the Protein Records Financial institution (6N7P for the UBC4 complex and 6N7R for the ACT1 complex). The cryo-EM maps were deposited in the Electron Microscopy Records Financial institution (EMD-0360 for the UBC4 complex and EMD-0361 for the ACT1 complex).

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Acknowledgements

This work used to be supported by NIH grants GM126157 and GM130673 (R.Z.); GM071940 and AI094386 (Z.H.Z.); and GM122579, GM121487, and CA219847 (R.D.). S.E. is a Howard Hughes Medical Institute Gilliam Fellow. K.K. used to be supported by an NSF GRFP award and a Stanford Graduate Fellowship. We acknowledge the exercise of instruments at the Electron Imaging Center for Nanomachines (supported by UCLA and by grants from the NIH (1S10OD018111, 1U24GM116792) and NSF (DBI-1338135 and DMR-1548924)) as well to the CU Anschutz Faculty of Remedy Cryo-EM and proteomics core facilities (partly supported by the Faculty of Remedy and the University of Colorado Cancer Center Enhance Grant P30CA046934). Molecular graphics and analyses were performed with the U.S. Chimera and ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with aid from NIGMS P41-GM103311 (Chimera, ChimeraX) and NIH R01-GM129325 (ChimeraX). We also thank M. Ares, D. Dark, and D. Foreheadfor feedback on early variations of the manuscript.

Author recordsdata

Author notes

  1. These authors contributed equally: Xueni Li, Shiheng Liu

Affiliations

  1. Department of Biochemistry and Molecular Genetics, Faculty of Remedy, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    • Xueni Li
    • , Lingdi Zhang
    • , Aaron Issaian
    • , Ryan C. Hill
    • , Sara Espinosa
    • , Shasha Shi
    • , Kirk C. Hansen
    •  & Rui Zhao
  2. Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA

    • Shiheng Liu
    •  & Z. Hong Zhou
  3. Electron Imaging Center for Nanomachines, UCLA, Los Angeles, CA, USA

    • Shiheng Liu
    • , Yanxiang Cui
    •  & Z. Hong Zhou
  4. Biophysics Program, Stanford University, Stanford, CA, USA

    • Kalli Kappel
    •  & Rhiju Das
  5. Department of Biochemistry, Stanford University, Stanford, CA, USA

    • Rhiju Das
  6. Department of Physics, Stanford University, Stanford, CA, USA

    • Rhiju Das
  7. RNA Bioscience Initiative, Faculty of Remedy, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    • Rui Zhao

Authors

  1. Seek Xueni Li in:

  2. Seek Shiheng Liu in:

  3. Seek Lingdi Zhang in:

  4. Seek Aaron Issaian in:

  5. Seek Ryan C. Hill in:

  6. Seek Sara Espinosa in:

  7. Seek Shasha Shi in:

  8. Seek Yanxiang Cui in:

  9. Seek Kalli Kappel in:

  10. Seek Rhiju Das in:

  11. Seek Kirk C. Hansen in:

  12. Seek Z. Hong Zhou in:

  13. Seek Rui Zhao in:

Contributions

X.L. and S.L. contributed equally to the work and are listed alphabetically in the author listing. R.Z. and Z.H.Z. conceived the mission; X.L. ready and optimized the sample; X.L., L.Z., S.E. and S.S. performed biochemical analyses; S.L. and Y.C. recorded and processed the EM recordsdata; A.I., R.C.H. and K.C.H. performed mass spectrometry analyses; S.L. constructed the atomic gadgets; K.K. and R.D. constructed the partial U1 snRNA model and the minimal exon model in the A posh; R.Z., S.L., X.L., and Z.H.Z. analysed and interpreted the gadgets; S.L., X.L. and R.Z. ready the illustrations; R.Z., S.L. and Z.H.Z. wrote the paper; and all authors contributed to the editing of the manuscript.

Corresponding authors

Correspondence to
Z. Hong Zhou or Rui Zhao.

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The authors hiss no competing pursuits.

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Extended recordsdata figures and tables

Extended Records Fig. 1 In vitro assembly and purification of the ACT1 complex.

a, Schematic illustration of the ACT1 pre-mRNA tagged with three MS2-binding sites (M3–ACT1) outdated-long-established for E complex assembly and purification. Boxes symbolize exon 1 (E1) and truncated exon 2 (E2). The 5′ SS (GU) and BPS (UACUAAC) are also confirmed. The red line represents the DNA oligo complementary to a local 5 nt upstream of the BPS for the RNase H cleavage experiment. b, RNA parts of the assembled E complex (with or without DNA oligo and RNase H treatment) after proteinase K digestion are confirmed on a denaturing urea gel or native agarose gel. These outcomes display that RNase treatment cleaved M3–ACT1 into two fragments. Present that the sizes of RNA on the native gel attach no longer match their linear dimension, per chance owing to the existence of secondary structures. This experiment used to be repeated two extra instances with identical outcomes.

Extended Records Fig. 2 The cryo-EM structural choice process for the ACT1 complex.

a, Consultant waft-corrected cryo-EM micrograph (out of 11,283 micrographs) of the E complex assembled on the ACT1 pre-mRNA. A representative particle is confirmed in a white dotted circle. b, Consultant 2D class averages of the ACT1 complex obtained in RELION. This experiment used to be repeated one beyond regular time with identical outcomes. c, Records processing workflow. For processing above the red dashed line, the particle photographs were binned to a pixel dimension of 2.72 Å. The relaxation of the processing used to be performed with a pixel dimension of 1.36 Å. The masks outdated-long-established in recordsdata processing are outlined with red accurate strains (peek Programs). d, Angular distribution of all particles outdated-long-established for the absolute most sensible 3.2 Å blueprint of the ACT1 complex. e, FSC as a feature of spatial frequency demonstrating the resolution of the absolute most sensible reconstruction of the ACT1 complex. f, Resmap local resolution estimation. g, FSC coefficients as a purposeful of spatial frequency between model and cryo-EM density maps. The on the total identical appearances between the FSC curves obtained with half of maps with (red) and without (blue) model refinement indicate that the refinement of the atomic coordinates did no longer undergo from excessive over-becoming.

Extended Records Fig. 3 The Cryo-EM structural choice process for the UBC4 complex.

a, Consultant waft-corrected cryo-EM micrograph (out of 8,997 micrographs) of the E complex assembled on the UBC4 pre-mRNA. A representative particle is confirmed in a white dotted circle. b, Consultant 2D class averages of the UBC4 complex obtained in RELION. c, Records processing workflow. For processing above the red dashed line, the particle photographs were binned to a pixel dimension of 2.72 Å. The relaxation of the processing used to be performed with a pixel dimension of 1.36 Å. The masks outdated-long-established in recordsdata processing are outlined with red accurate strains (peek Programs). d, Angular distribution of all particles outdated-long-established for the absolute most sensible 3.6 Å blueprint of the UBC4 complex. e, FSC as a feature of spatial frequency demonstrating the resolution of the absolute most sensible reconstruction of the UBC4 complex. f, Resmap local resolution estimation. g, FSC coefficients as a purposeful of spatial frequency between model and cryo-EM density maps. The on the total identical appearances between the FSC curves obtained with half of maps with (red) and without (blue) model refinement indicate that the refinement of the atomic coordinates did no longer undergo from excessive over-becoming.

Extended Records Fig. 4 Consultant cryo-EM density maps of the E complex.

ai, Densities for the UBC4 complex; j, density for the ACT1 complex. Cryo-EM density maps are confirmed as follows. a, Chosen areas of U1 snRNA. b, C-terminal space of Prp39. c, N-terminal domain of Snu71. d, Pre-mRNA and U1 snRNA duplex. e, U1C ZnF domain. f, Luc7 ZnF2 domain. g, Tandem FF domains of Prp40 (the identified structure of tandem FF domains from CA150 is also confirmed with the characteristic boomerang form). h, RRM2 domain of Nam8. i, NCBP1 and NCBP2. j, Broken-down density in the ACT1 complex that is assigned as the putative BBP–Mud2 heterodimer. The A posh is also confirmed, with U1 snRNP in the same orientation as the ACT1 complex and U2 snRNP located in identical positions as the BBP–Mud2 heterodimer with admire to U1 snRNP. The blueprint of the ACT1 complex used to be low-trail filtered to 40 Å.

Extended Records Fig. 5 Structural and biochemical characterization of the ACT1 and UBC4 complexes.

a, Comparability of the ribbon gadgets of the ACT1 complex, the UBC4 complex, and U1 snRNP from other beforehand sure structures (the U1 snRNP, A, and pre-B complexes). Labels with shading indicate protein or RNA parts that regulate between the ACT1 and UBC4 complexes. These parts and the RRM2 domain of Nam8 are also absent from beforehand sure structures. Present that U1–70K is shifted in the direction of NCBP2 in the UBC4 complex. b, Purified E complex does no longer hang U2 snRNA. A native polyacrylamide gel reveals the answer hybridization58 outcomes of total mobile RNA or RNA from purified E complex hybridized with fluorescent probes particular for U1 and U2 snRNAs. This experiment used to be repeated one beyond regular time with identical outcomes.

Extended Records Fig. 6 Secondary structures in the space between the 5′ SS and BPS in the wild-form and mutant ACT1 and UBC4 pre-mRNAs.

a, Secondary structures predicted by RNAstructure 6.0 (https://rna.urmc.rochester.edu/RNAstructureWeb/). b, Sequence between the 5′ SS and BPS (underlined) of ACT1. Purple nucleotides were mutated to A (as antagonistic to the one A, which used to be mutated to G) in mutant ACT1 to disrupt predicted secondary structures.

Extended Records Fig. 7 Protein interactions in the UBC4 complex.

a, DSSO crosslinking and mass spectrometry analyses of the UBC4 complex. Every blue line signifies a crosslink between a pair of Lys residues. Present that BBP–Mud2 are crosslinked to Luc7, Prp40, Snu56, and Snu71. b, Co-purification assays probing the interplay between Snu71 (or Prp40) and Luc7. Masses of combos of protein A–TEV–Prp40, protein A–TEV–Snu71, and CBP-tagged Luc7 or Luc7ΔCC (with coiled-coil domain (residues 123–190) deleted) were co-overexpressed in yeast (only Snu71 is protein A tagged in the Snu71 + Prp40 lanes), purified using IgG resin, eluted thru TEV cleavage, analysed on SDS–PAGE, and visualized using western blot with an anti-CBP antibody to detect Luc7 (high) and Ponceau S stain to display Snu71 or Prp40 (center). Western blot using the same anti-CBP antibody used to be outdated-long-established to display Luc7 expression ranges in cell lysates (bottom). The faint band spherical 26 kD in all lanes of the center gel is TEV. This experiment used to be repeated one beyond regular time with identical outcomes. c, The linker (residues 73–131) between the WW and FF domains of Prp40 is anticipated to be disordered using program MetaDisorderMD259.

Extended Records Fig. 8 Computational, biochemical, and structural characterization of the EDC.

a, The minimal dimension of RNA vital to connect the upstream division level (BP) and downstream 5′ SS in the A posh is modelled using the Rosetta RNP-denovo formula. The A posh (PDB ID 6G90) is confirmed in gray. The pre-mRNA is confirmed in inexperienced. The upstream division level and downstream 5′ SS are confirmed as red home-filling gadgets. Twenty-eight nucleotides are passable to connect the upstream division level and downstream 5′ SS (no longer together with the division level and 5′ SS themselves) without any chain spoil or clashes. b, Schematics of wild-form and mutant DYN2 pre-mRNA (mutated nucleotides confirmed in red), IEI, and untagged IEI outdated-long-established for the EDC assembly and in vivo exon definition experiments. Stem-loops symbolize the MS2 binding sites, and the red line represents the DNA oligonucleotide outdated-long-established for RNase H cleavage. c, SDS–PAGE reveals protein parts of complexes assembled on wild-form and IEI substrates (lanes 1, 2), on wild-form in the presence of competing untagged IEI (lane 3), and on IEI after RNase H treatment in the absence and presence of the DNA oligo (lanes 4, 5). This experiment used to be repeated one beyond regular time with identical outcomes. d, RNA parts of the same complexes as in lanes 4, 5 of c, confirming that RNase H treatment in the presence of the oligonucleotide cleaves the pre-mRNA. The smaller cleaved fragment (61 nucleotides) is sophisticated to scrutinize because EtBr stains short single-stranded RNA with low efficiency. This experiment used to be repeated two extra instances with identical outcomes. e, Mass spectrometry analyses of spliceosome assembled on the IEI and wild-form DYN2 pre-mRNA indicate that the two complexes secure the same parts in identical quantities with the exception of NCBP1 and a pair of, that are absent from the IEI complex. f, 2D classification of detrimental-stain TEM photographs of the E complex assembled on DYN2 IEI pre-mRNA. This experiment used to be repeated one beyond regular time with identical outcomes.

Extended Records Fig. 9 Characterization of circRNAs.

a, Sanger sequencing confirmed that the PCR merchandise in Fig. 5a were derived from T-branches and circRNAs of EFM5 and HMRA1. Solidus, space the put two ends of exon 2 are ligated; vertical line, space the put the 5′ SS of intron 2 is ligated to the BP of intron 1. The 5′ SS and BPS are confirmed in doughty. The BPS contains deletions (confirmed as -) as a result of errors triggered by reverse transcriptase reading thru the division. b, RT–PCR used to be utilized on RNA extracted from wild-form yeast cells with or without RNaseR treatment using primers indicated in the schematic diagrams beneath the gel, indicating that RNase R treatment eliminates linear RNAs. This experiment used to be repeated four extra instances with identical outcomes. c, Protein and RNA parts of E complex assembled on EFM5 IEI-101–M3 pre-mRNA. d, RT–PCR of RNA extracted from BY4742 yeast stress carrying indicated HRMA1 plasmids, with or without RNaseR treatment, using primers confirmed in the schematic diagrams beneath the gel. Numbers 246 and 62 designate exon lengths. Lanes 1–3 indicate that every body constructs were transcribed (endogenous HMRA1 pre-mRNA stage is simply too low to be detected as indicated in lane 3). The HMRA1 center exon used to be rather modified to waste a circRNA primer binding space so that only the modified exogenous (as an instance, IEI-246 in lane 5) nonetheless no longer wild-form HMRA1 circRNA (IEI-246 WT in lane 4) can be detected. e, IEI-246–M3 RNA or E complex assembled on IEI-246–M3 used to be incubated with wild-form or U1-depleted yeast extract in the absence or presence of 30-fold extra competing IEI-246 wild-form RNA. CircRNA merchandise were monitored using RT–PCR as in d. Experiments in ce were repeated one beyond regular time with identical outcomes.

Extended Records Desk 1 Cryo-EM recordsdata series, refinement and validation statistics.

Supplementary recordsdata

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