Pre-mRNA splicing in higher plants.

P re-mRNA splicing is one of the fundamental processes in constitutive and regulated gene expression in eukaryotes. During splicing, introns present in primary gene transcripts are removed and exons are ligated to produce translationally competent mRNAs. The basic mechanism of intron excision is similar in all eukaryotes. The reaction is mediated by the spliceosome, a large ribonucleoprotein (RNP) complex, which is assembled anew at each intron from small nuclear RNP particles (U-snRNPs) and numerous protein factors. Spliceosome assembly is a highly ordered and dynamic reaction, involving hydrolysis of several ATP molecules and many structural rearrangements Properties of plant introns The intron and exon organization of higher plant genes is similar to that of vertebrates In spite of these similarities, the requirements for intron recognition in plants differ from those in other eukaryotes, and plant cells generally fail to splice heterologous pre-mRNAs. The most important difference is a strong compositional bias for UA-or U-rich sequences in plant introns compared with those from yeast and vertebrates U12-type introns A minor class of nuclear pre-mRNA introns, referred to as U12-type or AT-AC introns (because they frequently start with AT and terminate with AC) have recently been described 3,13 . These introns contain different splice site and branch point sequences, and are excised by an alternative U12-type spliceosom

Role of Cajal Bodies and Nucleolus in the Maturation of the U1 snRNP in Arabidopsis

Background: The biogenesis of spliceosomal snRNPs takes place in both the cytoplasm where Sm core proteins are added and snRNAs are modified at the 59 and 39 termini and in the nucleus where snRNP-specific proteins associate. U1 snRNP consists of U1 snRNA, seven Sm proteins and three snRNP-specific proteins, U1-70K, U1A, and U1C. It has been shown previously that after import to the nucleus U2 and U4/U6 snRNP-specific proteins first appear in Cajal bodies (CB) and then in splicing speckles. In addition, in cells grown under normal conditions U2, U4, U5, and U6 snRNAs/snRNPs are abundant in CBs. Therefore, it has been proposed that the final assembly of these spliceosomal snRNPs takes place in this nuclear compartment. In contrast, U1 snRNA in both animal and plant cells has rarely been found in this nuclear compartment. Methodology/Principal Findings: Here, we analysed the subnuclear distribution of Arabidopsis U1 snRNP-specific proteins fused to GFP or mRFP in transiently transformed Arabidopsis protoplasts. Irrespective of the tag used, U1-70K was exclusively found in the nucleus, whereas U1A and U1C were equally distributed between the nucleus and the cytoplasm. In the nucleus all three proteins localised to CBs and nucleoli although to different extent. Interestingly, we also found that the appearance of the three proteins in nuclear speckles differ significantly. U1-70K was mostly found in speckles whereas U1A and U1C in,90 % of cells showed diffuse nucleoplasmic in combination with CBs and nucleolar localisation. Conclusions/Significance: Our data indicate that CBs and nucleolus are involved in the maturation of U1 snRNP. Difference

Use of Fluorescent Protein Tags to Study Nuclear Organization of the Spliceosomal Machinery in Transiently Transformed Living Plant Cells

Although early studies suggested that little compartmentalization exists within the nucleus, more recent studies on metazoan systems have identified a still increasing number of specific subnuclear compartments. Some of these compartments are dynamic structures; indeed, protein and RNA-protein components can cycle between different domains. This is particularly evident for RNA processing components. In plants, lack of tools has hampered studies on nuclear compartmentalization and dynamics of RNA processing components. Here, we show that transient expression of fluorescent protein fusions of U1 and U2 small nuclear ribonucleoprotein particle (snRNP)-specific proteins U1-70K, U2B″, and U2A ′, nucleolar proteins Nop10 and PRH75, and serine-arginine-rich proteins in plant protoplasts results in their correct localization. Furthermore, snRNP-specific proteins also were correctly assembled into mature snRNPs. This system allowed a systematic analysis of the cellular localization of Arabidopsis serine-arginine-rich proteins, which, like their animal counterparts, localize to speckles but not to nucleoli and Cajal bodies. Finally, markers for three different nuclear compartments, namely, nucleoli, Cajal bodies, and speckles, have been established and were shown to be applicable for colocalization studies in living plant protoplasts. Thus, transient expression of proteins tagged with four different fluorescent proteins is a suitable system for studying the nuclear organization of spliceosomal proteins in living plant cells and should therefore allow studies of their dynamics as well

AtCyp59 is a multidomain cyclophilin from Arabidopsis thaliana that interacts with SR proteins and the C-terminal domain of the RNA polymerase II

AtCyp59 and its orthologs from different organisms belong to a family of modular proteins consisting of a peptidyl-prolyl cis–trans isomerase (PPIase) domain, followed by an RNA recognition motif (RRM), and a C-terminal domain enriched in charged amino acids. AtCyp59 was identified in a yeast two-hybrid screen as an interacting partner of the Arabidopsis SR protein SCL33/SR33. The interaction with SCL33/SR33 and with a majority of Arabidopsis SR proteins was confirmed by in vitro pull-down assays. Consistent with these interactions, AtCyp59 localizes to the cell nucleus, but it does not significantly colocalize with SR proteins in nuclear speckles. Rather, it shows a punctuate localization pattern resembling transcription sites. Indeed, by using yeast two-hybrid, in vitro pull-down, and immunoprecipitation assays, we found that AtCyp59 interacts with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Ectopic expression of the tagged protein in Arabidopsis cell suspension resulted in highly reduced growth that is most probably due to reduced phosphorylation of the CTD. Together our data suggest a possible function of AtCyp59 in activities connecting transcription and pre-mRNA processing. We discuss our data in the context of a dynamic interplay between transcription and pre-mRNA processing

Localisation of transiently expressed U1 snRNP proteins in <i>Arabidopsis</i> protoplasts.

<p>(A) Single confocal sections of protoplasts expressing U1-70K, U1A, and U1C proteins fused to GFP. Corresponding differential interference contrast (DIC) image of a cell expressing U1-70K is also shown. Arrows, broken arrow and arrowheads point to nuclei, nucleoli and CBs, respectively. Scale bars, 15 µm. (B) Cellular localisation of GFP- (left panel) or HA-tagged (right panel) U1 snRNP-specific proteins studied by cellular fractionation. Cell extracts were fractionated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003989#pone.0003989-Lambermon1" target="_blank">[57]</a>. Lanes T, C, and N; total cellular, cytoplasmic, and nuclear protein fractions, respectively. Proteins were resolved by SDS-PAGE and analyzed by Western blotting, using mouse anti-GFP and rat anti-HA mAb. Molecular mass standards in kDa are indicated on the left. To control the quality of the fractionation procedure the same blots were probed with antibodies against nuclear and cytoplasmic proteins RBP45 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003989#pone.0003989-Lorkovic5" target="_blank">[58]</a> and fructose 1,6-bisphosphatase (cFBP), respectively (two bottom panels). (C) Immunodetection of U1-70K, U1A and U1C GFP fusion proteins in protein extract from transformed protoplasts. Total protein extracts were analysed by SDS-PAGE and Western blotting with anti-GFP antibody. Molecular mass standards in kDa are indicated on the left. Western blotting of the same protein extracts with anti-tubulin antibodies was performed as a loading control (bottom panel).</p

Co-localisation studies with U1 snRNP-specific proteins.

<p>(A) Co-localisation of U1A-GFP and U1C-mRFP. (B) Co-localisation of U1C-GFP and U2B″-mRFP. (C) Co-localisation of U170K-GFP and U1C-mRFP. Arrows, arrowheads, and asterisks point to nucleoli, CBs, and nucleolar cavities, respectively. All images are single confocal sections. Scale bars, 8 µm.</p

Nuclear distribution of transiently expressed U1-70K, U1A, and U1C proteins.

<p>Representative images of nuclear patterns observed in protoplasts expressing U1-70K (A), U1A (B), and U1C (C) GFP-tagged proteins. Single confocal sections are shown. Arrows, arrowheads, and asterisks point to nucleoli, CBs, and nucleolar cavities, respectively. Scale bars, 8 µm.</p

Transiently expressed U1 snRNP-specific proteins assemble into mature snRNP.

<p>(A) Immunoprecipitation of U1A-GFP and U1C-GFP fusion proteins with anti-m₃G antibody (α-m₃G). Lanes 1, input protein extract. Lanes 2, protein extracts incubated with protein-A Sepharose (pA). Lanes 3, immunoprecipitations with anti-m₃G antibody (α-m₃G). Arrowheads and arrows point to precipitated proteins and immunoglobulin heavy chains, respectively. The blot was probed with anti-GFP antibody. (B) Immunoprecipitation of U1A-HA and U1C-HA fusion proteins with anti-m₃G antibody (α-m₃G). The blots were probed with anti-HA antibody. Other details as in (A). (C) U1A-GFP and U1C-GFP fusion proteins precipitated with anti-GFP antibody co-immunoprecipitate U1 snRNAs. Left panel: lane 1, immunoprecipitation with anti-GFP antibody with protein extract from non-transformed protoplasts; lanes 2 and 5, input protein extract from cells expressing U1A-GFP and U1C-GFP fusion proteins, respectively; lanes 3 and 6, protein extracts from transformed cells incubated with protein-A Sepharose only (pA); lanes 4 and 7, immunoprecipitations with anti-GFP antibody (α-GFP) with protein extracts from transformed protoplasts. Arrowheads point to U1A and U1C GFP-tagged proteins and arrows point to immunoglobulin heavy and light chains. Right panel: analysis of anti-GFP immunoprecipitates (from the left panel, lanes 1, 4, and 7) for the presence of U1 snRNAs. After immunoprecipitation RNA was extracted, labelled by [³²P]-pCp ligation and analyzed on 8% denaturing PAA gels. Lane 1, RNA immunoprecipitated with anti-GFP antibody from non-transformed cells. Lanes 4 and 7, RNA co-precipitated with U1A-GFP and U1C-GFP, respectively.</p