1 / 72

Bio 525/ Spring, 2010 Nuclear Architecture and Genomic Function

Bio 525/ Spring, 2010 Nuclear Architecture and Genomic Function. Session 4: Background & Figures; Huang & Spector, 1996; Wei et al., 1999; Wei et al., 1998; Osborne et al., 2004;. Conventional View of Eukaryotic Transcription and RNA Splicing as two Separate Processes

keegan
Download Presentation

Bio 525/ Spring, 2010 Nuclear Architecture and Genomic Function

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Bio 525/ Spring, 2010Nuclear Architecture and Genomic Function Session 4: Background & Figures; Huang & Spector, 1996; Wei et al., 1999;Wei et al., 1998; Osborne et al., 2004;

  2. Conventional View of Eukaryotic Transcription • and RNA Splicing as two Separate Processes • DNA  Transcription  pre-mRNA • pre-mRNA RNA Splicing mRNA Current View of Eukaryotic Transcription and RNA Splicing as Being Functionally and Spatially Linked as a Highly Coordinated Process DNA Transcription/RNASplicing  mRNA

  3. HUANG S & SPECTOR DIntron-dependent recruitment of pre-mRNA splicing factors to sites of transcriptionJournal of Cell Biology (1996) 133, 719-732

  4. Huang & Spector , JCB 1996, Figure 1 Constructs used in transient transfections

  5. Major Conclusions of Huang & Spector, 1996 Conclusion 1 Association of nascent RNA transcripts with splicing factors is intron dependent during transient or stable expression (Figs 2-5).

  6. RNA SC-35 mergedHuang & Spector, Fig 2 β-galactosidase β-globin (cDNA) CMVTAT (partial intron) AD VA RNA Intronless RNA’s or RNA’s with a partial intron are not spatially associated with splicing factors in the cell nucleus

  7. RNA SC-35 merge Huang & Spector, Fig 3 β-globin (genomic DNA) CGTAT β- tropomyosin β-tropo/U2 snRNP Transiently expressed RNA transcripts containing introns are spatially associated with splicing factors

  8. Huang & Spector , JCB 1996, Figure 4 Association of transcripts and splicing factors

  9. Huang & Spector, Fig 5 RNA SC-35 merged β-globin minus β-globin plus Intron dependent association of RNA transcripts and splicing factors in stably transfected cell lines

  10. Major Conclusions of Huang & Spector , 1996 Conclusion 2 The foci of expressed RNA are also the sites of transcription (Fig 6).

  11. Huang & Spector, JCB 1996, Figure 6 RNA BrUTP merged β-globin minus β-tropo plus Transiently expressed RNAs co-localize with sites of transcription

  12. Major Conclusions of Huang & Spector , 1996 contd… Conclusion 3 The foci of intron-containing RNA are sites of pre-mRNA splicing and co-localize with mature mRNA (Fig 7 & 8).

  13. Huang & Spector , JCB 1996, Figure 7 Diagram illustrating the design of hybridization probes for the specific detection of β- tropomysin pre-mRNA and mature RNA. Introns 6 & 7 were used as pre-mRNA specific probes Splice junction constructs 12 Nt from 3’ and 5’ ends, respectively, between exons5 & 6 or 5 & 8 were used as mRNA specific probes

  14. Huang & Spector, JCB 1996, Figure 8 β-tropo mRNA (a) & pre-mRNA (b) β-tropo pre-mRNA (d) & SC-35 (e) β-tropo mRNA (g) & SC-35 (h) after 5 hr in α-amanitin Sites of transiently expressed intron-containing pre-mRNA & mRNA co-localize with each other and with sites of pre-mRNA splicing

  15. Major Conclusions of Huang & Spector, 1996 • Association of nascent RNA transcripts with splicing factors is intron dependent during transient or stable expression (Figs 2-5) • The foci of expressed RNA are also the sites of transcription (Fig 6) • The foci of intron-containing RNA are sites of pre-mRNA splicing and co-localize with mature mRNA (Fig 7 & 8) • Taken together these findings support a model for coordinate transcription and RNA splicing at discrete assemblies arranged within the nuclear architecture.

  16. A Model for Transcription-Coupled Pre-mRNA Splicing

  17. Ma H, SAMARABANDU J, DEVDHAR RS, ACHARYA R, CHENG P-C, MENG C & BEREZNEY R Spatial and temporal dynamics of DNA replication sites in mammalian cells. Journal of Cell Biology (1998) 143, 1415-1425. Review paper and powerpoint lecture 4 (Section II) from Bio 402/502 website

  18. 3-D Model of a 1 mbp Multi-Loop Chromatin Domain

  19. Functional Model of ~1 Mbp Chromatin Domains G1 Non-Replicating Chromatin Domain S Replication factory Replicating ChromatinDomain S or G2 Non-ReplicatingChromatin Domains

  20. DO THE HIGHER ORDER CHROMATIN DOMAINS (1 Mbp) FUNCTION AS GENE TRANSCRIPTION FACTORIES AND IF SO, WHAT IS THEIR RELATIONSHIP TO RNA SPLICING SITES (NUCLEAR SPECKLES)?

  21. Wei et al.Three-dimensional visualization of transcription sites and their association with splicing factor-rich nuclear specklesJournal of Cell Biology (1999) 146, 543-558

  22. Major Conclusions of Wei et al. , 1999 Conclusion 1 Transcription Sites (TS) labeled with BrUTP in permeabilized 3T3 mouse fibroblast cells are characterized by inhibitor and enzyme digestions studies as pol I- and II- RNAP mediated TS (Figure 1).

  23. Figure 1 (Wei et al., 1999): Properties of Transcription Sites in 3T3 Mouse Fibroblast Cells

  24. Major Conclusions of Wei et al. , 1999 Conclusion 2 Approx. 2,000 sites (94% pol II-mediated TS) are detected following segmentation and are arranged in clusters and 3-D network arrays (Figures 2 & 3).

  25. Figure 2 (Wei et al., 1999) Segmentation of Transcription Sites

  26. Figure 3 (Wei et al., 1999) 3-D Visualization of TS and Individual Sites

  27. Major Conclusions of Wei et al. , 1999 Conclusion 3 The number of TS, their 3-D organization and arrangement into higher order functional zones are maintained after extraction for nuclear matrix (Figures 4, 5 & 6).

  28. Figure 4 (Wei et al., 1999) TS After Nuclear Matrix Extraction and Synthesis on Nuclear Matrix After Nuclear Matrix Extraction On Nuclear Matrix

  29. Figure 5 (Wei et al., 1999) 3-D Visualization of TS and Individual Sites After Nuclear Matrix Extraction

  30. Figure 6 (Wei et al., 1999) Replication and Transcription Sites Higher Order Zones After Nuclear Matrix Extraction

  31. Major Conclusions of Wei et al. , 1999 Conclusion 4 Significant levels (43%) of pol II TS are associated with splicing factor-rich nuclear speckles (Figure 8, Table II) and are found along the periphery as well as inside individual speckles that are optically sectioned (Figure 9).

  32. Figure 8 (Wei et al., 1999) Association of TS With Nuclear Speckles

  33. Table II (Wei et al., 1999) Quantitation of TS Associated with Nuclear Speckles

  34. Figure 9 (Wei et al., 1999) Evaluation of TS Inside Speckles By Optical Sectioning

  35. Major Conclusions of Wei et al. , 1999 Conclusion 5 A similar distribution of TS along nuclear speckles was detected following in vivo labeling of TS with BrU (Figure 10).

  36. Figure 10 (Wei et al., 1999) In vivo Labeling of TS and Association With Nuclear Speckles

  37. Major Conclusions of Wei et al. , 1999 Overall Conclusion Taken together these findings support a model in which coordinate transcription/RNA splicing of pre-mRNA occurs in close association with the nuclear speckles as functional transcription/splicing factories that are associated with the nuclear matrix.

  38. Major Conclusions of Wei et al., 1999 • Transcription Sites (TS) labeled with BrdUTP in permeabilized 3T3 mouse fibroblast cells are characterized by inhibitor and enzyme digestions studies as pol I- and II- RNAP mediated TS (Figure 1). • Approx. 2,000 sites (94% pol II-mediated TS) are detected following segmentation and are arranged in clusters and 3-D network arrays (Figures 2 & 3). • The number of TS, their 3-D organization and arrangement into higher order functional zones are maintained after extraction for nuclear matrix (Figures 4,5 & 6). • Significant levels (43%) of pol II TS are associated with splicing factor-rich nuclear speckles (Figure 8, Table II) and are found along the periphery as well as inside individual speckles that are optically sectioned (Figure 9). • A similar distribution of TS along nuclear speckles was detected following in vivo labeling of TS with BrU (Figure 10). • Taken together these findings support a model in which coordinate transcription/RNA splicing of pre-mRNA occurs in close association with the nuclear speckles as functional transcription/splicing factories.

  39. Replication Timing Experiments in Mammalian Cells Have Demonstrate that Actively Transcribed Genes are Preferentially Replicated in Early S-Phase This raises the key question: When are the Genes in These Early S Replicated Chromatin Domains Transcribed?

  40. WEI X, SAMARABANDU J, DEVDHAR RS, SIEGEL AJ, ACHARYA R & BEREZNEY R Segregation of transcription and replication sites into higher order domains. Science (1998) 281, 1502-1505.

  41. RS TS RS/TS Simultaneous Replication and Transcription at Chromatin Domains during S-Phase

  42. Major Conclusions of Wei et al. , 1998 Conclusion 1 The ~ 1,000 RS & 2,000 TS at any moment in early S are spatially separate chromatin domains (Fig 1).

  43. Wei et al. , Science 1998, Figure 1 Spatial separation of replication and transcription sites throughout S phase

  44. These Findings Are Consistent With Model 2

  45. Major Conclusions of Wei et al. , 1998 contd.. Conclusion 2 Individual RS & TS are arranged into separate nuclear zones of replication or transcription (Fig 3).

  46. Wei et al. , Science 1998, Figure 3 Cluster distribution of replication and transcription sites extending over several optical sections

  47. These Findings Support A Novel Model 3

More Related