Wilhelm bernhard workshop on the cell nucleus

NUCLEAR TRANSLATION. AN OLD STORY

Iborra F.J. and Cook P.R.

Sir William Dunn School of Pathology, Oxford University, UK

It is widely believed that translation occurs only in the cytoplasm of eukaryotes, but our recent results suggest some takes place in nuclei coupled to transcription. The nonsense-mediated decay (NMD) pathway provides circumstantial evidence for this heterodoxy; it probably uses ribosomes to proofread messenger RNAs. We find components of the machineries involved in transcription, translation, and NMD colocalize, interact, and copurify, and that interactions between them are probably mediated by the C-terminal domain of the catalytic subunit of RNA polymerase II. These results can be explained if the NMD machinery uses nuclear ribosomes to translate – and so proofread – newly-made transcripts; then, faulty transcripts and any truncated peptides produced by nuclear translation would be degraded.

RECOGNITION OF OPEN READING FRAMES IN PRE-mRNAs WITHIN THE CELL NUCLEUS

Sperling R.¹ and Sperling J.²

¹The Hebrew University of Jerusalem, Jerusalem Israel; ²The Weizmann Institute of Science, Rehovot Israel
Pre-mRNA splicing involves recognition of a consensus sequence at the 5’ splice site. However, only some of the many potential sites that conform to the consensus are true ones, while the majority remain silent and are not normally used for splicing. We noticed that in most cases the utilization of such a latent intronic 5’splice site for splicing would introduce an in frame stop codon into the resultant mRNAs. This suggested a link between splice site selection and maintenance of an open reading frame, which is manifested by suppressing splicing events that could lead to the inclusion of pre-mature termination codons in mRNAs. We have experimentally verified this suppression of splicing (SOS) idea and found that splicing events at latent sites are indeed activated by mutations that eliminate the interfering stop codons. Attributing the null phenotype of latent splicing to nonsense-mediated mRNA decay (NMD) can be excluded because SOS, unlike NMD, is independent on translation. Nonetheless, latent splicing can be activated by eliminating start codons, indicating that at least start-codon-recognition is required for SOS. Our findings imply that the reading frame of mRNAs can be recognized in the cell nucleus prior to splicing, presumably by a surveillance mechanism that resides in the pre-mRNA processing machine. This nuclear scanning mechanism is highly important for accurate gene expression, as perturbations that would lead to splicing at latent sites are expected to introduce in frame stop codons into the majority of mRNAs.


EVIDENCES SUGGESTING THAT TRANS-CRIPTION IS INVOLVED IN HOMOLOGUE CHROMOSOME RECOGNITION AND ALIGNMENT PREVIOUS TO PAIRING IN EARLY MEIOTIC PROPHASE

Vázquez-Nin G.¹, Echeverría O.¹, Ortiz R.¹, Scassellati C.², Ubaldo E.¹, Fakan S.<sup>2

1</sup>Laboratory of Electron Microscopy, Department of Biology, Faculty of Sciences, National Autonomous University of Mexico (UNAM), Mexico; ²Center of Electron Microscopy, University of Lausanne, Switzerland
The nuclei of guinea pig spermatogonia and spermatocytes were studied by means of electron microscopic methods such as high resolution cytochemistry, immunocytochemistry, in situ hybridization, and quantitative autoradiography at light microscope level. Immunolocalization of DNA, RNA polymerase II, and hnRNPs suggest that filaments of extended chromatin resembling very small lampbrush structures and pairing elements transcribe hnRNA intensively. The immunocytochemical localization of

hnRNPs, snRNPs, and the trimethyl-guanosin cap of snRNAs demonstrates that splicing is scarce in the nucleus of spermatocytes in early meiotic prophase, in which recognition, alignment and pairing is taking place. The results of quantitative autoradiography after ³H-uridine labeling, show an intense transcription accompanied by a very slow export of RNA to the cytoplasm. These results lead us to propose that newly transcribed mRNA stays long time in the nucleus, or never leaves it, to fulfill a function related to homologous searching, recognition and alignment at molecular level, previous to pairing.

ß-Actin, TAta binding protein (TBP) and transcription by RNA polymerase II (RNAPII)

de Lanerolle P., Mavrommatis E., Johnson, T., Hoffman W., Stojiljkovic L., Fuchsova B., Philimonenko V.¹ and Hozak P.¹

Department of Physiology, University of Illinois at Chicago, Chicago, USA and ¹Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Actin is abundant in the nucleus and actin is reported to co-purify with RNAPII (Egly et al., 1984). We have also demonstrated that a new isoform of myosin I in the nucleus (Pestic-Dragovic et al., 2000). Nuclear myosin I (NMI) co-localizes with RNA polymerase II (RNAPII) and antibodies to NMI inhibit transcription by RNAPII. We now report that the microinjection of antibodies to NMI or to ß-actin into the nuclei of HeLa cells block transcription, in vivo. Assays using purified RNAPII, recombinant TBP, TFIIB and TFIIF and negatively supercoiled DNA showed that antibodies to NMI or to ß-actin inhibited transcription, in vitro, while control antibodies did not. We have also found ß-actin in pre-initiation complexes (PIC). Furthermore, TBP co-IP's with ß-actin and vice versa in the presence or absence of sarcosyl. Other experiments have shown that ß-actin co-localizes with TBP. Binding studies using peptides derived from ß-actin have shown that certain peptides inhibit the ß-actin-TBP interaction while other peptides do not. Based on these data, we propose ß-actin is involved in PIC formation through an interaction with TBP and that ß-actin and NMI act as molecular motors to power transcription by RNAPII.

References:

Egly et al., EMBO J. , 3: 2363-71, 1984

Pestic-Dragovic et al., 2000