DOI: 10.1073/pnas.95.8.4188. The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices<i>in vitro</i>

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplicesin vitro

10.1073/pnas.95.8.4188

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplicesin vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Bibliography

Laggerbauer, B., Achsel, T., & LÃ¼hrmann, R. (1998). The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplicesin vitro. Proceedings of the National Academy of Sciences, 95(8), 4188â4192.

Authors 3

Bernhard Laggerbauer (first)
Tilmann Achsel (additional)
Reinhard Lührmann (additional)

References 37 Referenced 172

M J Moore, C C Query, P A Sharp The RNA World, eds R F Gesteland, J F Atkins (Cold Spring Harbor Lab. Press, Plainview, NY), pp. 303–357 (1993). / The RNA World by Moore M J (1993)
10.1016/S0955-0674(97)80003-8
10.1016/0092-8674(94)90563-0
10.1146/annurev.ge.28.120194.000245
10.1016/0092-8674(93)90531-T
10.1111/j.1365-2958.1992.tb01470.x
10.1016/S0959-440X(05)80116-2
10.1038/349463a0
10.1007/978-3-662-22325-3_5
10.1002/yea.320020105
10.1073/pnas.87.11.4236
10.1101/gad.1.5.445
10.1038/349487a0
10.1101/gad.5.4.629
10.1073/pnas.94.22.11798
10.1128/mcb.14.11.7611-7620.1994
S Teigelkamp, C Mundt, T Achsel, C Will, R Lührmann RNA 3, 1313–1326 (1997). / RNA by Teigelkamp S (1997)
10.1073/pnas.94.22.11803
10.1101/gad.11.14.1864
10.1002/j.1460-2075.1992.tb05291.x
10.1074/jbc.271.52.33261
10.1038/349494a0
10.1002/j.1460-2075.1992.tb05610.x
10.1128/MCB.16.12.6810
10.1002/j.1460-2075.1996.tb00774.x
10.1093/genetics/143.1.67
10.1038/381709a0
J Lin, J Y Rossi RNA 2, 835–848 (1996). / RNA by Lin J (1996)
10.1093/nar/24.5.868
C L Will, B Kastner, R Lührmann RNA Processing, eds S J Higgins, B D Hames (IRL, Oxford) II, 141–177 (1994). (10.1093/oso/9780199633449.003.0005) / RNA Processing by Will C L (1994)
10.1101/gad.3.12b.2137
10.1016/0092-8674(89)90334-6
10.1002/j.1460-2075.1986.tb04447.x
J Lauber, G Plessel, S Prehn, C L Will, P Fabrizio, K Gröning, W S Lane, R Lührmann RNA 3, 926–941 (1997). / RNA by Lauber J (1997)
10.1038/334213a0
D A Brow, R M Vidaver RNA 1, 122–131 (1995). / RNA by Brow D A (1995)
10.1093/emboj/16.13.4092

Dates

Type	When
Created	23 years ago (July 26, 2002, 10:40 a.m.)
Deposited	1 year, 7 months ago (Jan. 4, 2024, 12:04 a.m.)
Indexed	2 weeks, 3 days ago (Aug. 6, 2025, 8:08 a.m.)
Issued	27 years, 4 months ago (April 14, 1998)
Published	27 years, 4 months ago (April 14, 1998)
Published Online	27 years, 4 months ago (April 14, 1998)
Published Print	27 years, 4 months ago (April 14, 1998)

Funders 0

None

BibTeX

@article{Laggerbauer_1998, title={The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplicesin vitro}, volume={95}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.95.8.4188}, DOI={10.1073/pnas.95.8.4188}, number={8}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Laggerbauer, Bernhard and Achsel, Tilmann and Lührmann, Reinhard}, year={1998}, month=apr, pages={4188–4192} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        8,
        6
      ]
    ],
    "date-time": "2025-08-06T12:08:37Z",
    "timestamp": 1754482117781
  },
  "reference-count": 37,
  "publisher": "Proceedings of the National Academy of Sciences",
  "issue": "8",
  "content-domain": {
    "domain": [
      "www.pnas.org"
    ],
    "crossmark-restriction": true
  },
  "published-print": {
    "date-parts": [
      [
        1998,
        4,
        14
      ]
    ]
  },
  "abstract": "<jats:p>Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA\u2013RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5\u2013200kD and U5\u2013100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices<jats:italic>in vitro</jats:italic>. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5\u2013200kD protein but lack U5\u2013100kD, suggesting that the U5\u2013200kD protein could mediate U4/U6 duplex unwinding. Finally, U5\u2013200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5\u2013200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.</jats:p>",
  "DOI": "10.1073/pnas.95.8.4188",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2002,
        7,
        26
      ]
    ],
    "date-time": "2002-07-26T14:40:52Z",
    "timestamp": 1027694452000
  },
  "page": "4188-4192",
  "update-policy": "http://dx.doi.org/10.1073/pnas.cm10313",
  "source": "Crossref",
  "is-referenced-by-count": 172,
  "title": "The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices<i>in vitro</i>",
  "prefix": "10.1073",
  "volume": "95",
  "author": [
    {
      "given": "Bernhard",
      "family": "Laggerbauer",
      "sequence": "first",
      "affiliation": [
        {
          "name": "Institut f\u00fcr Molekularbiologie und Tumorforschung, Philipps-Universit\u00e4t Marburg, Emil-Mannkopff-Stra\u03b2e 2, D-35037 Marburg, Germany"
        }
      ]
    },
    {
      "given": "Tilmann",
      "family": "Achsel",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Institut f\u00fcr Molekularbiologie und Tumorforschung, Philipps-Universit\u00e4t Marburg, Emil-Mannkopff-Stra\u03b2e 2, D-35037 Marburg, Germany"
        }
      ]
    },
    {
      "given": "Reinhard",
      "family": "L\u00fchrmann",
      "sequence": "additional",
      "affiliation": [
        {
          "name": "Institut f\u00fcr Molekularbiologie und Tumorforschung, Philipps-Universit\u00e4t Marburg, Emil-Mannkopff-Stra\u03b2e 2, D-35037 Marburg, Germany"
        }
      ]
    }
  ],
  "member": "341",
  "published-online": {
    "date-parts": [
      [
        1998,
        4,
        14
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_3_3_1_2",
      "first-page": "303",
      "volume-title": "The RNA World",
      "author": "Moore M J",
      "year": "1993",
      "unstructured": "M J Moore, C C Query, P A Sharp The RNA World, eds R F Gesteland, J F Atkins (Cold Spring Harbor Lab. Press, Plainview, NY), pp. 303\u2013357 (1993)."
    },
    {
      "key": "e_1_3_3_2_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0955-0674(97)80003-8"
    },
    {
      "key": "e_1_3_3_3_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(94)90563-0"
    },
    {
      "key": "e_1_3_3_4_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.ge.28.120194.000245"
    },
    {
      "key": "e_1_3_3_5_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(93)90531-T"
    },
    {
      "key": "e_1_3_3_6_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1365-2958.1992.tb01470.x"
    },
    {
      "key": "e_1_3_3_7_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0959-440X(05)80116-2"
    },
    {
      "key": "e_1_3_3_8_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/349463a0"
    },
    {
      "key": "e_1_3_3_9_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1007/978-3-662-22325-3_5"
    },
    {
      "key": "e_1_3_3_10_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/yea.320020105"
    },
    {
      "key": "e_1_3_3_11_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.87.11.4236"
    },
    {
      "key": "e_1_3_3_12_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1101/gad.1.5.445"
    },
    {
      "key": "e_1_3_3_13_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/349487a0"
    },
    {
      "key": "e_1_3_3_14_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1101/gad.5.4.629"
    },
    {
      "key": "e_1_3_3_15_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.94.22.11798"
    },
    {
      "key": "e_1_3_3_16_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/mcb.14.11.7611-7620.1994"
    },
    {
      "key": "e_1_3_3_17_2",
      "first-page": "1313",
      "volume": "3",
      "author": "Teigelkamp S",
      "year": "1997",
      "unstructured": "S Teigelkamp, C Mundt, T Achsel, C Will, R L\u00fchrmann RNA 3, 1313\u20131326 (1997).",
      "journal-title": "RNA"
    },
    {
      "key": "e_1_3_3_18_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.94.22.11803"
    },
    {
      "key": "e_1_3_3_19_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1101/gad.11.14.1864"
    },
    {
      "key": "e_1_3_3_20_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1992.tb05291.x"
    },
    {
      "key": "e_1_3_3_21_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.271.52.33261"
    },
    {
      "key": "e_1_3_3_22_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/349494a0"
    },
    {
      "key": "e_1_3_3_23_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1992.tb05610.x"
    },
    {
      "key": "e_1_3_3_24_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/MCB.16.12.6810"
    },
    {
      "key": "e_1_3_3_25_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1996.tb00774.x"
    },
    {
      "key": "e_1_3_3_26_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/genetics/143.1.67"
    },
    {
      "key": "e_1_3_3_27_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/381709a0"
    },
    {
      "key": "e_1_3_3_28_2",
      "first-page": "835",
      "volume": "2",
      "author": "Lin J",
      "year": "1996",
      "unstructured": "J Lin, J Y Rossi RNA 2, 835\u2013848 (1996).",
      "journal-title": "RNA"
    },
    {
      "key": "e_1_3_3_29_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/24.5.868"
    },
    {
      "key": "e_1_3_3_30_2",
      "doi-asserted-by": "crossref",
      "first-page": "141",
      "DOI": "10.1093/oso/9780199633449.003.0005",
      "volume-title": "RNA Processing",
      "author": "Will C L",
      "year": "1994",
      "unstructured": "C L Will, B Kastner, R L\u00fchrmann RNA Processing, eds S J Higgins, B D Hames (IRL, Oxford) II, 141\u2013177 (1994)."
    },
    {
      "key": "e_1_3_3_31_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1101/gad.3.12b.2137"
    },
    {
      "key": "e_1_3_3_32_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0092-8674(89)90334-6"
    },
    {
      "key": "e_1_3_3_33_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1986.tb04447.x"
    },
    {
      "key": "e_1_3_3_34_2",
      "first-page": "926",
      "volume": "3",
      "author": "Lauber J",
      "year": "1997",
      "unstructured": "J Lauber, G Plessel, S Prehn, C L Will, P Fabrizio, K Gr\u00f6ning, W S Lane, R L\u00fchrmann RNA 3, 926\u2013941 (1997).",
      "journal-title": "RNA"
    },
    {
      "key": "e_1_3_3_35_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/334213a0"
    },
    {
      "key": "e_1_3_3_36_2",
      "first-page": "122",
      "volume": "1",
      "author": "Brow D A",
      "year": "1995",
      "unstructured": "D A Brow, R M Vidaver RNA 1, 122\u2013131 (1995).",
      "journal-title": "RNA"
    },
    {
      "key": "e_1_3_3_37_2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/emboj/16.13.4092"
    }
  ],
  "container-title": "Proceedings of the National Academy of Sciences",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://pnas.org/doi/pdf/10.1073/pnas.95.8.4188",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2024,
        1,
        4
      ]
    ],
    "date-time": "2024-01-04T05:04:34Z",
    "timestamp": 1704344674000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://pnas.org/doi/full/10.1073/pnas.95.8.4188"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1998,
        4,
        14
      ]
    ]
  },
  "references-count": 37,
  "journal-issue": {
    "issue": "8",
    "published-print": {
      "date-parts": [
        [
          1998,
          4,
          14
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1073/pnas.95.8.4188"
  ],
  "URL": "http://dx.doi.org/10.1073/pnas.95.8.4188",
  "relation": {},
  "ISSN": [
    "0027-8424",
    "1091-6490"
  ],
  "subject": [],
  "container-title-short": "Proc. Natl. Acad. Sci. U.S.A.",
  "published": {
    "date-parts": [
      [
        1998,
        4,
        14
      ]
    ]
  },
  "assertion": [
    {
      "value": "1997-12-01",
      "order": 0,
      "name": "received",
      "label": "Received",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "1998-01-12",
      "order": 1,
      "name": "accepted",
      "label": "Accepted",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    },
    {
      "value": "1998-04-14",
      "order": 2,
      "name": "published",
      "label": "Published",
      "group": {
        "name": "publication_history",
        "label": "Publication History"
      }
    }
  ]
}