No abstract

Transcription and splicing must proceed over genomic distances of hundreds of kilobases in many human genes. However, the rates and mechanisms of these processes are poorly understood. We have used the compound 5,6-Dichlorobenzimidazole 1-b-D-ribofuranoside (DRB) that reversibly blocks gene transcription in vivo combined with quantitative RT-PCR to analyze the transcription and RNA processing of several long human genes. We found that the rate of RNA polymerase II transcription over long genomic distances is about 3.8 kb per minute and is nearly the same whether transcribing long introns or exon rich regions. We also determined that co-transcriptional pre-mRNA splicing of U2-dependent introns occurs within 5–10 minutes of synthesis irrespective of intron length between 1 kb and 240 kb. Similarly, U12-dependent introns were co-transcriptionally spliced within 10 minutes of synthesis confirming that these introns are spliced within the nuclear compartment. These results show that the expression of large genes is surprisingly rapid and efficient.

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.

U2-type and U12-type introns are spliced by distinct spliceosomes in eukaryotic nuclei. A classification method was devised to distinguish these two types of introns based on splice site sequence properties and was used to identify 56 different genes containing U12-type introns in available genomic sequences. U12-type introns occur with consistently low frequency in diverse eukaryotic taxa but have almost certainly been lost from C. elegans. Comparisons with available homologous sequences demonstrate subtype switching of U12 introns between termini of AT-AC and GT-AG as well as conversion of introns from U12-type to U2-type and provide evidence for a fission/fusion model in which the two splicing systems evolved in separate lineages that were fused in a eukaryotic progenitor.

Summary Core RNA processing reactions in eukaryotic cells occur cotranscriptionally in a chromatin context, but the relationship between chromatin structure and pre-mRNA processing is poorly understood. We observed strong nucleosome depletion around human polyadenylation sites (PAS), and nucleosome enrichment just downstream of PAS. In genes with multiple alternative PAS, higher downstream nucleosome affinity was associated with higher PAS usage, independently of known PAS motifs that function at the RNA level. Conversely, exons were associated with distinct peaks in nucleosome density. Exons flanked by long introns or weak splice sites exhibited stronger nucleosome enrichment, and incorporation of nucleosome density data improved splicing simulation accuracy. Certain histone modifications, including H3K36me3 and H3K27me2, were specifically enriched on exons, suggesting active marking of exon locations at the chromatin level. Together, these findings provide evidence for extensive functional connections between chromatin structure and RNA processing.