Electron Tomography Reveals Posttranscriptional Binding of Pre-Mrnps to Specific Fibers in the Nucleoplasm

Using electron tomography, we have analyzed whether the Balbiani ring (BR) pre-mRNP particles in transit from the gene to the nuclear pore complex (NPC) are bound to any structure that could impair free diffusion through the nucleoplasm. We show that one-third of the BR particles are in contact with thin connecting fibers (CFs), which in some cases merge into large fibrogranular clusters. The CFs have a specific protein composition different from that of BR particles, as shown by immuno-EM. Moreover, we have identified hrp65 as one of the protein components of the CFs. The sequencing of hrp65 cDNA reveals similarities with hnRNP proteins and splicing factors. However, hrp65 is likely to have a different function because it does not bind to nascent pre-mRNA and is not part of the pre-mRNP itself. Taken together, our observations indicate that pre-mRNPs are not always freely diffusible in the nucleoplasm but interact with fibers of specific structure and composition, which implies that some of the posttranscriptional events that the pre-mRNPs undergo before reaching the NPC occur in a bound state

Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions

Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3′ end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes. Thus, individual endogenous pre-mRNPs/mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo

Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation

A combination of several rate-limiting steps allows for efficient control of alternative splicing

The hrp23 Protein in the Balbiani Ring Pre-mRNP Particles Is Released Just before or at the Binding of the Particles to the Nuclear Pore Complex

Balbiani ring (BR) pre-mRNP particles reside in the nuclei of salivary glands of the dipteran Chironomus tentans and carry the message for giant-sized salivary proteins. In the present study, we identify and characterize a new protein component in the BR ribonucleoprotein (RNP) particles, designated hrp23. The protein with a molecular mass of 20 kD has a single RNA-binding domain and a glycine-arginine-serine–rich auxiliary domain. As shown by immunoelectron microscopy, the hrp23 protein is added to the BR transcript concomitant with transcription, is still present in the BR particles in the nucleoplasm, but is absent from the BR particles that are bound to the nuclear pore complex or are translocating through the central channel of the complex. Thus, hrp23 is released just before or at the binding of the particles to the nuclear pore complex. It is noted that hrp23 behaves differently from two other BR RNP proteins earlier studied: hrp36 and hrp45. These proteins both reach the nuclear pore complex, and hrp36 even accompanies the RNA into the cytoplasm. It is concluded that each BR RNA-binding protein seems to have a specific flow pattern, probably related to the particular role of the protein in gene expression

The translational landscape of the splicing factor SRSF1 and its role in mitosis

The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer.Fil: Maslon, Magdalena M. . Institute of Genetics and Molecular Medicine, University of Edinburgh; Reino UnidoFil: Heras, Sara R.. Institute of Genetics and Molecular Medicine, University of Edinburgh; Reino Unido. Universidad de Granada; EspañaFil: Bellora, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación En Biodiversidad y Medioambiente; Argentina. Universitat Pompeu Fabra; EspañaFil: Eyras, Eduardo. Universitat Pompeu Fabra; España. Catalan Institution for Research and Advanced Studies (ICREA), Barcelona; EspañaFil: Cáceres, Javier F.. University Of Edinburgh; Reino Unid

Pre-mRNA processing in the polytene nuclei of chironomus tentans

PRE-mRNA PROCESSING IN THE POLYTENE NUCLEI OF CHIRONOMUS TENTANS Göran Bauren Department of Cell and Molecular Biology, The medical Nobel Institute Karolinska Institute, S-171 77 Stockholm Gene expression of protein encoding genes in eukaryotic cells requires extensive intranuclear maturation processing of the transcribed pre-mRNA. Three such processes are known to occur. The 5' end is capped and the 3' end is determined by cleavage and polyadenylation. A majority of pre-mRNA transcripts also contain intervening sequences, introns, that have to be removed before transport to the cytoplasm. All three processing events need protein or protein/RNA complexes. Biochemical assays in vertebrate systems and genetic analysis in yeast have identified a majority of the. factors involved in the processes and the biochemical pathway of each reaction has also been studied in some detail. Much less is known about the different processes in the intact cell nucleus, and their relationship to each other and transcription. The aim of this thesis was to study the cell biology of the above mentioned maturation processing events in terms of functional organization of the different processing machineries in relation to each other, and to the organization of the transcription machinery in the intact nucleus of polytene Chironomus tentans cells. Polytene nuclei were studied as they provide exceptional experimental possibilities compared to diploid cells We showed that splicing factors and functional splicesomes are associated with the growing pre-mRNA transcript, and that splicing could occur on nascent transcripts. One major factor determining if an intron will be removed co- or posttranscriptionally is its location in the pre-mRNA molecule, 5' located introns are removed cotranscriptionally more frequent than 3' located ones. We also demonstrated that there is a linear polarity between the location of an intron and its probability to be removed. This is not true for closely spaced introns where factors like intron size and splice site sequences play a more predomunant role. By using antibodies detecting different splicing components we demonstrated a dynamic organization of splicing factors in the cell nucleus. The majority of the different factors are located in the nucleoplasm, 80-90%, while the remaining part is associated with nascent pre-mRNA molecules. There is a dramatic reorganization of splicing factors upon transcriptional inhibition. Splicing factors that were bound to nascent pre-mRNA leave the chromosomes and are concentrated in the nucleoplasm. The opposite was recorded when activation of specific genes were performed. Termination of BRl gene transcription occur in a region 600 bases downstream the polyadenylation site, and termination is in close relation to cleavage and polyadenylation. A small population of polyadenylated molecules could be found in the BR1 locus. These molecules have removed the final intron to a high degree (80%) compared to the nascent molecules (5-10%). In addition to this their poly(A) tail is short, only 20 adenosine residues compared to 100 residues in the nucleoplasm, indicating a slow initial phase of the addition of adenosines. Key words: Pre-mRNA processing, Organization of splicing factors, Cotranscriptional splicing, Transcription termination, Chironomus tentans, Polytene nuclei. ISBN 91-628-2242-