Publicação
Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity
| Resumo: | Removal of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20-30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min(-1) and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context. |
|---|---|
| Autores principais: | Martin, Robert M. |
| Outros Autores: | Rino, José; Carvalho, Célia; Kirchhausen, Tomas; Carmo-Fonseca, Maria |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | artigo |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| _version_ | 1866811094697246720 |
|---|---|
| author | Martin, Robert M. |
| author2 | Rino, José Carvalho, Célia Kirchhausen, Tomas Carmo-Fonseca, Maria |
| author2_role | author author author author |
| author_facet | Martin, Robert M. Rino, José Carvalho, Célia Kirchhausen, Tomas Carmo-Fonseca, Maria |
| author_role | author |
| contributor_name_str_mv | Repositório Científico de Acesso Aberto da ULisboa |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Martin, Robert M.\",\"Person.identifier.orcid\":\"0000-0001-5600-7525\"},{\"Person.name\":\"Rino, José\",\"Person.identifier.orcid\":\"0000-0002-0637-4527\"},{\"Person.name\":\"Carvalho, Célia\",\"Person.identifier.orcid\":\"0000-0002-5980-595X\"},{\"Person.name\":\"Kirchhausen, Tomas\"},{\"Person.name\":\"Carmo-Fonseca, Maria\",\"Person.identifier.orcid\":\"0000-0002-3402-7143\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Repositório Científico de Acesso Aberto da ULisboa |
| datacite.creators.creator.creatorName.fl_str_mv | Martin, Robert M. Rino, José Carvalho, Célia Kirchhausen, Tomas Carmo-Fonseca, Maria |
| datacite.date.Accepted.fl_str_mv | 2013-01-01T00:00:00Z |
| datacite.date.available.fl_str_mv | 2021-12-03T15:47:59Z |
| datacite.date.embargoed.fl_str_mv | 2021-12-03T15:47:59Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| datacite.titles.title.fl_str_mv | Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity |
| dc.contributor.none.fl_str_mv | Repositório Científico de Acesso Aberto da ULisboa |
| dc.creator.none.fl_str_mv | Martin, Robert M. Rino, José Carvalho, Célia Kirchhausen, Tomas Carmo-Fonseca, Maria |
| dc.date.Accepted.fl_str_mv | 2013-01-01T00:00:00Z |
| dc.date.available.fl_str_mv | 2021-12-03T15:47:59Z |
| dc.date.embargoed.fl_str_mv | 2021-12-03T15:47:59Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | http://hdl.handle.net/10451/50271 |
| dc.language.none.fl_str_mv | eng |
| dc.publisher.none.fl_str_mv | Elsevier |
| dc.rights.cclincense.fl_str_mv | http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| dc.title.fl_str_mv | Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_6501 |
| description | Removal of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20-30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min(-1) and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context. |
| dirty | 0 |
| eu_rights_str_mv | openAccess |
| format | article |
| fulltext.url.fl_str_mv | https://repositorio.ulisboa.pt/bitstreams/89050d87-edce-4cff-9c64-ce570af648f4/download |
| funding.funder.alternateName_str_mv | FCT FCT |
| funding.funder.identifier_str_mv | http://doi.org/10.13039/501100001871 http://doi.org/10.13039/501100001871 |
| funding.funder.name_str_mv | Fundação para a Ciência e a Tecnologia Fundação para a Ciência e a Tecnologia |
| funding.name_str_mv | 3599-PPCDT |
| id | ul_f92e89bba1323ef5cc4e10d00124cffb |
| identifier.url.fl_str_mv | http://hdl.handle.net/10451/50271 |
| instacron_str | ul |
| institution | Universidade de Lisboa |
| instname_str | Universidade de Lisboa |
| language | eng |
| network_acronym_str | ul |
| network_name_str | Repositório da Universidade de Lisboa |
| oai_identifier_str | oai:repositorio.ulisboa.pt:10451/50271 |
| organization_str_mv | urn:organizationAcronym:ul |
| person_str_mv | Martin, Robert M. Martin, Robert M. https://www.ciencia-id.pt/7516-6C51-460B 7516-6C51-460B http://orcid.org/0000-0001-5600-7525 0000-0001-5600-7525 Rino, José Rino, José https://www.ciencia-id.pt/A117-FBDB-A27B A117-FBDB-A27B http://orcid.org/0000-0002-0637-4527 0000-0002-0637-4527 Carvalho, Célia Carvalho, Célia https://www.ciencia-id.pt/DD18-667D-59FE DD18-667D-59FE http://orcid.org/0000-0002-5980-595X 0000-0002-5980-595X Kirchhausen, Tomas Carmo-Fonseca, Maria Carmo-Fonseca, Maria https://www.ciencia-id.pt/B31F-0435-0753 B31F-0435-0753 http://orcid.org/0000-0002-3402-7143 0000-0002-3402-7143 |
| publishDate | 2013 |
| publisher.none.fl_str_mv | Elsevier |
| reponame_str | Repositório da Universidade de Lisboa |
| repository_id_str | urn:repositoryAcronym:ul |
| service_str_mv | urn:repositoryAcronym:ul |
| spelling | engElsevierpt_PTRemoval of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20-30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min(-1) and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.application/pdfpt_PTLive-cell visualization of pre-mRNA splicing with single-molecule sensitivityPersonalMartin, Robert M.DSpacehttp://dspace.org/items/d50e7a9a-5216-4738-a5c6-94c8c0b85a1dDSpacehttp://dspace.org/items/d50e7a9a-5216-4738-a5c6-94c8c0b85a1dMartinRobert ManfredCiência IDhttps://www.ciencia-id.pt7516-6C51-460BORCIDhttp://orcid.org0000-0001-5600-7525PersonalRino, JoséDSpacehttp://dspace.org/items/54f3b487-a7a1-4597-aa2e-65f660855937DSpacehttp://dspace.org/items/54f3b487-a7a1-4597-aa2e-65f660855937Rino HenriquesJosé MiguelCiência IDhttps://www.ciencia-id.ptA117-FBDB-A27BORCIDhttp://orcid.org0000-0002-0637-4527PersonalCarvalho, CéliaDSpacehttp://dspace.org/items/0bc60872-4f9a-44d0-9bd6-13c82c3298c2DSpacehttp://dspace.org/items/0bc60872-4f9a-44d0-9bd6-13c82c3298c2CarvalhoCéliaCiência IDhttps://www.ciencia-id.ptDD18-667D-59FEORCIDhttp://orcid.org0000-0002-5980-595XKirchhausen, TomasPersonalCarmo-Fonseca, MariaDSpacehttp://dspace.org/items/f4cae50c-2389-4b6a-8e3a-8fd8e538add0DSpacehttp://dspace.org/items/f4cae50c-2389-4b6a-8e3a-8fd8e538add0Carmo-FonsecaMariaCiência IDhttps://www.ciencia-id.ptB31F-0435-0753ORCIDhttp://orcid.org0000-0002-3402-7143Scopus Author IDhttps://www.scopus.com7007128195HostingInstitutionOrganizationalRepositório Científico de Acesso Aberto da ULisboae-mailmailto:repositorio@reitoria.ulisboa.ptrepositorio@reitoria.ulisboa.ptISSNIsPartOf2211-1247DOIIsPartOf10.1016/j.celrep.2013.08.0132021-12-03T15:47:59Z20132013-01-01T00:00:00ZHandlehttp://hdl.handle.net/10451/50271http://purl.org/coar/access_right/c_abf2open access3725795 bytesFundação para a Ciência e a TecnologiaSplicing and stochasticity in programmed cell death decisions3599-PPCDTCrossref Funder IDhttp://doi.org/10.13039/501100001871Fundação para a Ciência e a TecnologiaA SYNTHETIC BIOLOGY APPROACH TO STUDY SPLICING REGULATIONCrossref Funder IDhttp://doi.org/10.13039/501100001871literaturehttp://purl.org/coar/resource_type/c_6501journal article2013http://creativecommons.org/licenses/by-nc-nd/4.0/http://purl.org/coar/access_right/c_abf2application/pdffulltexthttps://repositorio.ulisboa.pt/bitstreams/89050d87-edce-4cff-9c64-ce570af648f4/downloadCell Reports4611441155 |
| spellingShingle | Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity Martin, Robert M. |
| status | SINGLETON |
| title | Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity |
| title_full | Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity |
| title_fullStr | Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity |
| title_full_unstemmed | Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity |
| title_short | Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity |
| title_sort | Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity |
| url | http://hdl.handle.net/10451/50271 |
| visible | 1 |