Richard A. Padgett

Splicing of Messenger RNA Precursors

A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted. This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns. The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome. This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6. A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome.

Biased chromatin signatures around polyadenylation sites and exons.

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.

Rates of in situ transcription and splicing in large human genes

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-β-D-ribofuranoside (DRB), which 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 min−1 and is similar 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 min of synthesis, irrespective of intron length between 1 kb and 240 kb. Similarly, U12-dependent introns were co-transcriptionally spliced within 10 min of synthesis, confirming that these introns are spliced within the nuclear compartment. These results show that the expression of large genes is unexpectedly rapid and efficient.

Mutations in the spliceosome machinery, a novel and ubiquitous pathway in leukemogenesis

Myelodysplastic syndromes (MDSs) are chronic and often progressive myeloid neoplasms associated with remarkable heterogeneity in the histomorphology and clinical course. Various somatic mutations are involved in the pathogenesis of MDS. Recently, mutations in a gene encoding a spliceosomal protein, SF3B1, were discovered in a distinct form of MDS with ring sideroblasts. Whole exome sequencing of 15 patients with myeloid neoplasms was performed, and somatic mutations in spliceosomal genes were identified. Sanger sequencing of 310 patients was performed to assess phenotype/genotype associations. To determine the functional effect of spliceosomal mutations, we evaluated pre-mRNA splicing profiles by RNA deep sequencing. We identified additional somatic mutations in spliceosomal genes, including SF3B1, U2AF1, and SRSF2. These mutations alter pre-mRNA splicing patterns. SF3B1 mutations are prevalent in low-risk MDS with ring sideroblasts, whereas U2AF1 and SRSF2 mutations are frequent in chronic myelomonocytic leukemia and advanced forms of MDS. SF3B1 mutations are associated with a favorable prognosis, whereas U2AF1 and SRSF2 mutations are predictive for shorter survival. Mutations affecting spliceosomal genes that result in defective splicing are a new leukemogenic pathway. Spliceosomal genes are probably tumor suppressors, and their mutations may constitute diagnostic biomarkers that could potentially serve as therapeutic targets.

Evolutionary Fates and Origins of U12-Type Introns

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.

Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein

A mouse monoclonal antibody and human autoimmune sera directed against various classes of small ribonucleoprotein particles have been tested for inhibition of mRNA splicing in a soluble in vitro system. The splicing of the first and second leader exons of adenovirus late RNA was inhibited only by those sera that reacted with U1 RNP. Both U1 RNP-specific human autoimmune serum and sera directed against the Sm class of small nuclear RNPs, including a mouse monoclonal antibody, specifically inhibited splicing. Antisera specific for U2 RNP had no effect on splicing nor did antisera specific for the La or Ro class of small RNPs. These results suggest that U1 RNP is essential for the splicing of mRNA precursors.

Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns

A conserved sequence element in a minor class of eukaryotic pre-messenger RNA (pre-mRNA) introns was previously proposed to base pair with a complementary sequence in the U12 small nuclear RNA (snRNA) in a manner analogous to the pairing of U2 snRNA with the branch site sequence of the major class of introns. Here, mutations generated in this conserved sequence element block the splicing of a member of this minor intron class in vivo. This block was relieved by coexpression of a U12 snRNA containing compensatory mutations that restore the proposed base pairing interaction. These results show that this minor class of pre-mRNA introns is a distinct class existing alongside the major class of introns in animal genomes, and these results also establish an in vivo function for U12 snRNA.

Mutations in U4atac snRNA, a Component of the Minor Spliceosome, in the Developmental Disorder MOPD I

Minor RNA splicing defects can cause a major human developmental disorder. Small nuclear RNAs (snRNAs) are essential factors in messenger RNA splicing. By means of homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays showed that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns were found to be poorly spliced in MOPD I patient fibroblast cells. The introduction of wild-type U4atac snRNA into MOPD I cells enhanced U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development.

SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

New connections between splicing and human disease

The removal by splicing of introns from the primary transcripts of most mammalian genes is an essential step in gene expression. Splicing is performed by large, complex ribonucleoprotein particles termed spliceosomes. Mammals contain two types that splice out mutually exclusive types of introns. However, the role of the minor spliceosome has been poorly studied. Recent reports have now shown that mutations in one minor spliceosomal snRNA, U4atac, are linked to a rare autosomal recessive developmental defect. In addition, very exciting recent results of exome deep-sequencing have found that recurrent, somatic, heterozygous mutations of other splicing factors occur at high frequencies in particular cancers and pre-cancerous conditions, suggesting that alterations in the core splicing machinery can contribute to tumorigenesis. Mis-splicing of crucial genes may underlie the pathologies of all of these diseases. Identifying these genes and understanding the mechanisms involved in their mis-splicing may lead to advancements in diagnosis and treatment.