Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells.

Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5' untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing.

How slow RNA polymerase II elongation favors alternative exon skipping.

Splicing is functionally coupled to transcription, linking the rate of RNA polymerase II (Pol II) elongation and the ability of splicing factors to recognize splice sites (ss) of various strengths. In most cases, slow Pol II elongation allows weak splice sites to be recognized, leading to higher inclusion of alternative exons. Using CFTR alternative exon 9 (E9) as a model, we show here that slowing down elongation can also cause exon skipping by promoting the recruitment of the negative factor ETR-3 onto the UG-repeat at E9 3' splice site, which displaces the constitutive splicing factor U2AF65 from the overlapping polypyrimidine tract. Weakening of E9 5' ss increases ETR-3 binding at the 3' ss and subsequent E9 skipping, whereas strengthening of the 5' ss usage has the opposite effect. This indicates that a delay in the cotranscriptional emergence of the 5' ss promotes ETR-3 recruitment and subsequent inhibition of E9 inclusion.

Transcriptional elongation and alternative splicing.

Alternative splicing has emerged as a key contributor to proteome diversity, highlighting the importance of understanding its regulation. In recent years it became apparent that splicing is predominantly cotranscriptional, allowing for crosstalk between these two nuclear processes. We discuss some of the links between transcription and splicing, with special emphasis on the role played by transcription elongation in the regulation of alternative splicing events and in particular the kinetic model of alternative splicing regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

First come, first served revisited: factors affecting the same alternative splicing event have different effects on the relative rates of intron removal.

Alternative splicing accounts for much of the complexity in higher eukaryotes. Thus, its regulation must allow for flexibility without hampering either its specificity or its fidelity. The mechanisms involved in alternative splicing regulation, especially those acting through coupling with transcription, have not been deeply studied in in vivo models. Much of our knowledge comes from in vitro approaches, where conditions can be precisely controlled at the expense of losing several levels of regulation present in intact cells. Here we studied the relative order of removal of the introns flanking a model alternative cassette exon. We show that there is a preferential removal of the intron downstream from the cassette exon before the upstream intron has been removed. Most importantly, both cis-acting mutations and trans-acting factors that regulate the model alternative splicing event differentially affect the relative order of removal. However, reduction of transcriptional elongation causing higher inclusion of the cassette exon does not change the order of intron removal, suggesting that the assumption, according to the "first come, first served" model, that slow elongation promotes preferential excision of the upstream intron has to be revised. We propose instead that slow elongation favors commitment to exon inclusion during spliceosome assembly. Our results reveal that measuring the order of intron removal may be a straightforward read-out to discriminate among different mechanisms of alternative splice site selection.

Strong polyadenylation and weak pausing combine to cause efficient termination of transcription in the human Ggamma-globin gene.

The human gamma-globin genes form part of a 5-kb tandem duplication within the beta-globin gene cluster on chromosome 11. Despite a high degree of identity between the two genes, we show that while the upstream Ggamma-globin gene terminates transcription efficiently, termination in the Agamma gene is inefficient. This is primarily due to the different strengths of the polyA signals of the two genes; Ggamma-globin has a functionally stronger polyA signal than the Agamma gene. The probable cause of this difference in polyA efficiency characteristics lies with a number of base changes which reduce the G/U content of the GU/U-rich region of the Agamma polyA signal relative to that of Ggamma. The 3' flanking regions of the two gamma-globin genes have similar abilities to promote transcription termination. We found no evidence to suggest a cotranscriptional cleavage event, such as that seen in the human beta-globin gene, occurs in either gamma-globin 3' flank. Instead we find evidence that the 3' flank of the Ggamma-globin gene contains multiple weak pause elements which, combined with the strong polyA signal the gene possesses, are likely to cause gradual termination across the 3' flank.