Célia Carvalho

The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive.

fAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.

Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity

Removal of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20-30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min(-1) and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.

Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes

In the nucleus of higher eukaryotes, maturation of mRNA precursors involves an orderly sequence of transcription-coupled interdependent steps. Transcription is well known to influence splicing, but how splicing may affect transcription remains unclear. Here we show that a splicing mutation that prevents recruitment of spliceosomal snRNPs to nascent transcripts causes co-transcriptional retention of unprocessed RNAs that remain associated with polymerases stalled predominantly at the 3′ end of the gene. In contrast, treatment with spliceostatin A, which allows early spliceosome formation but destabilizes subsequent assembly of the catalytic complex, abolishes 3′ end pausing of polymerases and induces leakage of unspliced transcripts to the nucleoplasm. Taken together, the data suggest that recruitment of splicing factors and correct assembly of the spliceosome are coupled to transcription termination, and this might ensure a proofreading mechanism that slows down release of unprocessed transcripts from the transcription site.

Depletion of the yeast nuclear exosome subunit Rrp6 results in accumulation of polyadenylated RNAs in a discrete domain within the nucleolus

ABSTRACT Recent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)+ RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)+ RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome.

In situ visualisation of immunoglobulin genes in normal and malignant lymphoid cells

Aims—To directly visualise immunoglobulin (Ig) heavy (H) and light chain genes (κ and λ) in metaphase chromosomes and interphase nuclei of normal and malignant lymphocytes using small genomic probes targeted to intragenic sequences. Methods—Cytogenetic preparations from phytohaemagglutinin stimulated lymphocytes, B-chronic lymphocytic leukaemia (B-CLL) cells, and a B-prolymphocytic leukaemia (B-PLL) cell line, containing a t(11;14), were hybridised in situ using biotin or digoxigenin labelled plasmid probes. The κ genes were visualised with a combination of probes for the Cκ, Jκ, Vκ1, and Vκ2 segments, the λ genes with a probe containing the Jλ2-Cλ2, Jλ3-Cλ3 segments and the H genes with a probe for Cλ2. Hybridisation sites were visualised using appropriate fluorochrome conjugates and images were analysed by digital microscopy. Results—In both normal and malignant lymphoid cells, the κ and λ genes were visualised as a single dot signal, whereas the H λ genes were resolved as either two or three separate signals per chromatid in metaphase chromosomes or per allele in interphase nuclei. In the malignant PLL cells, double hybridisation experiments with a painting library specific for the chromosome 11 showed that the λ region was retained in the translocated chromosome, with an in situ resolution pattern similar to that of the normal allele. Conclusions—This study shows that a high resolution in situ analysis of the three Ig loci can be efficiently performed with small size genomic probes on both normal and malignant lymphoid cells. Such an approach offers a flexible tool for the molecular characterisations of these loci on chromosomes and individual neoplastic cells.

influence of transcription and replication on the in situ resolution of immunoglobulin heavy-chain constant region genes: an interphase cytogenetics analysis

An interphase cytogenetics analysis was performed to investigate whether replication and transcription could influence in situ resolution of immunoglobulin (Ig) heavy chain constant region genes. A plasmid probe recognizing five Cγ segments separated by known linear DNA distances was hybridized in situ and visualized by digital fluorescence microscopy. In interphase nuclei from phytohemagglutinin (PHA)-stimulated lymphocytes, the γ genes were resolved as one to three signals per allele in the majority of nuclei, whereas in a minority, complex patterns of several signals per allele could be observed. The latter were restricted to nuclei in an early stage of the S phase, as assessed by hybridization experiments performed in cells grown in the presence of bromodeoxyuridine. To investigate whether the in situ resolution of the Cγ segments could vary as a function of the transcription activity of the locus, the Cγ probe was subsequently hybridized to nuclei from a mature B cell line (JVM-2), which produces γ transcripts as shown by in situ RNA hybridization experiments. Primary human fibroblasts were further used as representative of a non-lymphoid cell type with transcriptionally inactive Ig genes. When Gl nuclei from the three cell types were compared in terms of the in situ resolution of the Cγ locus, JVM-2 cells were found to include the highest percentage of higher resolution patterns (three to five signals per allele in 28% of nuclei), fibroblasts the lowest (three signals per allele, 2%), while PHA-stimulated lymphocytes occupied an intermediate position between the other two cell types (three or four signals per allele, 15%). The data show that the in situ resolution of Ig Cγ genes varies throughout the cell cycle and is influenced by the transcriptional activity of the locus. The variability of the resolution patterns observed appears to reflect different levels of chromatin packaging, which in turn are likely to influence the probe accessibility to its target. These observations are relevant for the interpretation of data from interphase cytogenetics analysis of independent, but closely spaced, DNA segments.

Single-Molecule Imaging of RNA Splicing in Live Cells

Expression of genetic information in eukaryotes involves a series of interconnected processes that ultimately determine the quality and amount of proteins in the cell. Many individual steps in gene expression are kinetically coupled, but tools are lacking to determine how temporal relationships between chemical reactions contribute to the output of the final gene product. Here, we describe a strategy that permits direct measurements of intron dynamics in single pre-mRNA molecules in live cells. This approach reveals that splicing can occur much faster than previously proposed and opens new avenues for studying how kinetic mechanisms impact on RNA biogenesis.

Short (16-mer) locked nucleic acid splice-switching oligonucleotides restore dystrophin production in Duchenne Muscular Dystrophy myotubes

Splice-switching antisense oligonucleotides (SSOs) offer great potential for RNA-targeting therapies, and two SSO drugs have been recently approved for treating Duchenne Muscular Dystrophy (DMD) and Spinal Muscular Atrophy (SMA). Despite promising results, new developments are still needed for more efficient chemistries and delivery systems. Locked nucleic acid (LNA) is a chemically modified nucleic acid that presents several attractive properties, such as high melting temperature when bound to RNA, potent biological activity, high stability and low toxicity in vivo. Here, we designed a series of LNA-based SSOs complementary to two sequences of the human dystrophin exon 51 that are most evolutionary conserved and evaluated their ability to induce exon skipping upon transfection into myoblasts derived from a DMD patient. We show that 16-mers with 60% of LNA modification efficiently induce exon skipping and restore synthesis of a truncated dystrophin isoform that localizes to the plasma membrane of patient-derived myotubes differentiated in culture. In sum, this study underscores the value of short LNA-modified SSOs for therapeutic applications.

In Situ Hybridization for Simultaneous Detection of DNA, RNA, and Protein

Publisher Summary Fluorescence in situ hybridization enables the detection of RNA and DNA in the cellular context, therefore allowing the study of gene expression at the single cell level. The use of different probe labels and detection systems with multiple fluorochromes made possible the simultaneous detection of several target sequences in the same cell, including the detection of a specific gene and the corresponding pre-mRNA and/or mRNA. A major problem when conjugating protocols to simultaneously detect DNA, RNA, and proteins is to achieve good preservation of the different targets throughout the procedure and, at the same time, make them accessible for the probes. There are several methods to label DNA probes. Choosing a specific method depends on the kind of probe to be labeled. The oligonucleotide probes should be at least 40–50 nucleotides long to minimize the possibility of unspecific binding to nontarget cellular RNAs. When choosing the sequences, avoid regions that may form highly stable secondary structures or dimmerise.