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.

FAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.

Studies over the past years indicate that there is extensive coupling between nuclear export of mRNA and pre-mRNA processing. Here, we visualized the distribution of exon junction complex (EJC) proteins and RNA export factors relative to sites of abundant pre-mRNA synthesis in the nucleus. We analyzed both HeLa cells infected with adenovirus and murine erythroleukemia (MEL) cells stably transfected with the human β-globin gene. Using in situ hybridization and confocal microscopy, we observe accumulation of EJC proteins (REF/Aly, Y14, SRm160, UAP56, RNPS1, and Magoh) and core spliceosome components (U snRNPs) at sites of transcription. This suggests that EJC proteins bind stably to pre-mRNA cotranscriptionally. No concentration of the export factors NXF1/TAP, p15, and Dbp5 was detected on nascent transcripts, arguing that in mammalian cells these proteins bind the mRNA shortly before or after release from the sites of transcription. These results also suggest that binding of EJC proteins to the mRNA is not sufficient to recruit TAP-p15, consistent with recent findings showing that the EJC does not play a crucial role in mRNA export. Contrasting to the results obtained in MEL cells expressing normal human β-globin transcripts, mutant pre-mRNAs defective in splicing and 3′end processing do not colocalize with SRm160, REF, UAP56, or Sm proteins. This shows that the accumulation of EJC proteins at transcription sites requires efficient processing of the nascent pre-mRNAs, arguing that transcription per se is not sufficient for the stable assembly of the EJC.

Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric α-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or throughtrans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric α-satellite DNA in human lymphoid cells synchronized at G₀/G₁is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric α-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that “chromosomal environment” plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.

Recent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)⁺ RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)⁺ RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome.