Further ultrastructural characterization of the intranuclear ring-shaped bodies of the plant Lacandonia schismatica

Ring-shaped bodies are found in the nucleus of Lacandonia schismatica, a rare plant with the sexual organs inverted. They are 0.5-microm-diameter structures that present an electron-dense external ring surrounding a central core. Ultrastructural studies indicate that these bodies contain RNA. The external ring is labeled with antibodies against small nuclear ribonucleoproteins, suggesting that they may be involved in pre-mRNA metabolism. In the present work we further characterized these intranuclear ring-shaped structures by serial-sectioning analysis. Moreover, we tested the presence of additional molecular elements related to pre-mRNA metabolism, such as SR proteins and poly(A)(+) RNA, using immunoelectron microscopy and ultrastructural in situ hybridization. Our results show that these nuclear bodies are spherical. They contain SR proteins involved in splicing and postsplicing events and little to no poly(A)(+) RNA. We also found similar nuclear bodies in other plant and animal species. Therefore, ring-shaped bodies in L. schismatica are spherical, highly compartmentalized nuclear structures that may be involved in pre-mRNA metabolism.     

Cajal bodies in insect oocytes. I. Identification and immunocytochemical characteristics of Cajal bodies in vitellogenic oocytes of the yellow mealworm beetle

In vitellogenic oocytes of Tenebrio molitor (inactive stage), numerous fibrogranular nuclear bodies (NBs) are present. Using immunofluorescent microscopy, these NBs were shown to contain pre-mRNA splicing factors (small nuclear [sn] RNPs and SR-protein, SC35) as well as RNA polymerase II. A limited set of NBs also contained coilin, a marker protein for Cajal bodies (CBs). We suggest that in T. molitor oocytes, coilin-containing NBs, which also contain splicing factors and RNA polymerase II, seem to represent CBs. In the species studied, no morphological features of CBs were established as compared with other NBs, which do not contain coilin. Microinjectons in oocytes of myc-tagged coilin mRNA, followed by revealing newly translated protein with antibody specific for this tag, have shown targeting of myc-coilin with CBs. The own and literary data on the morphology and molecular composition of CBs are discussed in terms of searching for criteria for CB identification in cells of different origin, and at active and inactive stages.     

In vitro splicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA

The human calcitonin/CGRP-I (CALC-I) gene can be alternatively expressed into calcitonin mRNA in thyroid C-cells and into CGRP-I mRNA in particular nerve cells. Formation of calcitonin mRNA requires splicing of exons 1, 2, 3 and 4 and addition of poly(A) at exon 4, whereas splicing of exons 1, 2, 3, 5 and 6 and addition of poly(A) at exon 6 yields CGRP-I mRNA. The calcitonin and CGRP-I mRNA-specific splicing reactions were investigated in vitro, in nuclear extracts of HeLa cells, using model precursor RNAs containing the exon 3 to exon 5 region of the gene. A precursor RNA containing the full-length exon 3 to exon 5 region was only poorly spliced in vitro. Therefore, a systematic analysis was performed of the effect of deletions introduced in the intron 3, exon 4 and intron 4 of this precursor RNA on calcitonin/CGRP mRNA-specific splicing. The deletions increased the efficiency of splicing considerably. In all cases CGRP mRNA-specific splicing is strongly favoured over calcitonin mRNA-specific splicing. In addition, splicing reactions using cryptic 5' splice sites were detected which interfered with the usage of processing signals for calcitonin and CGRP mRNA-specific splicing. The results imply a major regulatory role for the exon 4 poly(A) addition reaction in the generation of calcitonin mRNA.     

Solution structure of the HIV-1 exon splicing silencer 3

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impair HIV-1 replication. Genome splicing in HIV-1 is tightly regulated by the dynamic assembly/disassembly of trans host factors with cis RNA control elements. The host protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, regulates splicing at several highly conserved HIV-1 3′ splice sites by binding 5′-UAG-3′ elements embedded within regions containing RNA structure. The physical determinants of hnRNP A1 splice site recognition remain poorly defined in HIV-1, thus precluding a detailed understanding of the molecular basis of the splicing pattern. Here, the three-dimensional structure of the exon splicing silencer 3 (ESS3) from HIV-1 has been determined using NMR spectroscopy. ESS3 adopts a 27-nucleotide hairpin with a 10-bp A-form stem that contains a pH-sensitive A⁺C wobble pair. The seven-nucleotide hairpin loop contains the high-affinity hnRNP-A1-responsive 5′-UAGU-3′ element and a proximal 5′-GAU-3′ motif. The NMR structure shows that the heptaloop adopts a well-organized conformation stabilized primarily by base stacking interactions reminiscent of a U-turn. The apex of the loop is quasi-symmetric with UA dinucleotide steps from the 5′-GAU-3′ and 5′-UAGU-3′ motifs stacking on opposite sides of the hairpin. As a step towards understanding the binding mechanism, we performed calorimetric and NMR titrations of several hnRNP A1 subdomains into ESS3. The data show that the UP1 domain forms a high-affinity (K_d = 37.8 ± 1.1 nM) complex with ESS3 via site-specific interactions with the loop.