Most plant primary transcripts undergo alternative splicing (AS), and its impact on protein diversity is a subject of intensive investigation. Several studies have uncovered various mechanisms of how particular protein splice isoforms operate. However, the common principles behind the AS effects on protein function in plants have rarely been surveyed. Here, on the selected examples, we highlight diverse tissue expression patterns, subcellular localization, enzymatic activities, abilities to bind other molecules and other relevant features. We describe how the protein isoforms mutually interact to underline their intriguing roles in altering the functionality of protein complexes. Moreover, we also discuss the known cases when these interactions have been placed inside the autoregulatory loops. This review is particularly intended for plant cell and developmental biologists who would like to gain inspiration on how the splice variants encoded by their genes of interest may coordinately work.

African trypanosomes are early divergent protozoan parasites responsible for high mortality and morbidity as well as a great economic burden among the world's poorest populations. Trypanosomes undergo antigenic variation in their mammalian hosts, a highly sophisticated immune evasion mechanism. Their nuclear organization and mechanisms for gene expression control present several conventional features but also a number of striking differences to the mammalian counterparts. Some of these unorthodox characteristics, such as lack of controlled transcription initiation or enhancer sequences, render their monogenic antigen transcription, which is critical for successful antigenic variation, even more enigmatic. Recent technological developments have advanced our understanding of nuclear organization and gene expression control in trypanosomes, opening novel research avenues. This review is focused on Trypanosoma brucei nuclear organization and how it impacts gene expression, with an emphasis on antigen expression. It highlights several dedicated sub-nuclear bodies that compartmentalize specific functions, whilst outlining similarities and differences to more complex eukaryotes. Notably, understanding the mechanisms underpinning antigen as well as general gene expression control is of great importance, as it might help designing effective control strategies against these organisms.

RNase MRP is a ribonucleoprotein enzyme involved in processing precursor rRNA in eukaryotes. To facilitate our structure–function analysis of RNase MRP from Saccharomyces cerevisiae, we have determined the likely secondary structure of the RNA component by a phylogenetic approach in which we sequenced all or part of the RNase MRP RNAs from 17 additional species of the Saccharomycetaceae family. The structure deduced from these sequences contains the helices previously suggested to be common to the RNA subunit of RNase MRP and the related RNA subunit of RNase P, an enzyme cleaving tRNA precursors. However, outside this common region, the structure of RNase MRP RNA determined here differs from a previously proposed universal structure for RNase MRPs. Chemical and enzymatic structure probing analyses were consistent with our revised secondary structure. Comparison of all known RNase MRP RNA sequences revealed three regions with highly conserved nucleotides. Two of these regions are part of a helix implicated in RNA catalysis in RNase P, suggesting that RNase MRP may cleave rRNA using a similar catalytic mechanism.

The Pescadillo protein was identified via a developmental defect and implicated in cell cycle progression. Here we report that human Pescadillo and its yeast homolog (Yph1p or Nop7p) are localized to the nucleolus. Depletion of Nop7p leads to nuclear accumulation of pre-60S particles, indicating a defect in subunit export, and it interacts genetically with a tagged form of the ribosomal protein Rpl25p, consistent with a role in subunit assembly. Two pre-rRNA processing pathways generate alternative forms of the 5.8S rRNA, designated 5.8SL and 5.8SS. In cells depleted for Nop7p, the 27SA3 pre-rRNA accumulated, whereas later processing intermediates and the mature 5.8SS rRNA were depleted. Less depletion was seen for the 5.8SL pathway. TAP-tagged Nop7p coprecipitated precursors to both 5.8SL and 5.8SS but not the mature rRNAs. We conclude that Nop7p is required for efficient exonucleolytic processing of the 27SA3 pre-rRNA and has additional functions in 60S subunit assembly and transport. Nop7p is a component of at least three different pre-60S particles, and we propose that it carries out distinct functions in each of these complexes.

Conversion of tRNA precursors to their mature forms requires the action of both endo- and exoribonucleases. Although studies over many years identified the endoribonuclease, RNase P, and several exoribonucleases as the enzymes responsible for generating the mature 5′ and 3′ termini, respectively, of Escherichia coli tRNAs, relatively little is known about how tRNAs are separated from long multimeric or multifunction transcripts, or from long leader and trailer sequences. To examine this question, the tRNA products that accumulate in mutant strains devoid of multiple exoribonucleases plus one or several endoribonucleases were analyzed by northern analysis. We find that the multifunction tyrT transcript, which contains two tRNA1Tyr sequences separated by a 209-nt spacer region plus a downstream mRNA, is cleaved at three sites in the spacer region by the endoribonuclease, RNase E. When both RNase E and RNase P are absent, a product containing both tRNAs accumulates. Two multimeric tRNA transcripts, those for tRNA Arg-His-Leu-Pro and tRNA Gly-Cys-Leu also require RNase E for maturation. For the former transcript, products with long 3′ extensions on tRNAArg, tRNAHis, and tRNAPro, as well as the primary transcript, accumulate in the absence of RNase E. For the latter transcript, RNase E cleaves downstream of each tRNA. Little processing of either multimeric transcript occurs in the absence of both RNase E and RNase P. These data indicate that RNase E is a major contributor to the initial processing of E. coli tRNA transcripts, providing substrates for final maturation by RNase P and the 3′ exoribonucleases. Based on this new information, a detailed model for tRNA maturation is proposed.

The U3 small nucleolar ribonucleoprotein (snoRNP) is composed of a small nucleolar RNA (snoRNA) and at least 10 proteins. The U3 snoRNA base pairs with the pre-rRNA to carry out the A0, A1, and A2 processing reactions that lead to the release of the 18S rRNA from the nascent pre-rRNA transcript. The yeast U3 snoRNA can be divided into a short 5′ domain (nt 1–39) and a larger 3′ domain (73 to the 3′ end) separated by a stretch of nucleotides called the hinge region (nt 40–72). The sequences required for pre-rRNA base pairing are found in the 5′ domain and hinge region whereas the 3′ domain is largely covered with proteins. Mpp10p, one of the protein components unique to the U3 snoRNP, plays a role in processing at the A1 and A2 sites. Because of its critical role in U3 snoRNP function, we determined which sequences in the U3 snoRNA are required for Mpp10p association. Unlike fibrillarin and all the previous U3 snoRNP components studied in this manner, sequences in the 3′ domain are not sufficient for Mpp10p association. Instead, a conserved sequence element in the U3 snoRNA hinge region is required, placing Mpp10p near the 5′ domain that carries out the pre-rRNA base-pairing interactions in the functional center of the U3 snoRNP.

About half of Caenorhabditis elegans genes have a 1–2 bp mismatch to the canonical AAUAAA hexamer that signals 3′ end formation. One rare variant, AGUAAA, is found at the 3′ end of the mai-1 gene, the first gene in an operon also containing gpd-2 and gpd-3. When we expressed this operon under heat shock control, 3′ end formation dependent on the AGUAAA was very inefficient, but could be rescued by a single bp change to create a perfect AAUAAA. When AGUAAA was present, most 3′ ends formed at a different site, 100 bp farther downstream, right at the gpd-2 trans-splice site. Surprisingly, 3′ end formation at this site did not require any observable match to the AAUAAA consensus. It is possible that 3′ end formation at this site occurs by a novel mechanism—trans-splicing-dependent cleavage—as deletion of the trans-splice site prevented 3′ end formation here. Changing the AGUAAA to AAUAAA also influenced the trans-splicing process: with AGUAAA, most of the gpd-2 product was trans-spliced to SL1, rather than SL2, which is normally used at downstream operon trans-splice sites. However, with AAUAAA, SL2 trans-splicing of gpd-2 was increased. Our results imply that (1) the AAUAAA consensus controls 3′ end formation frequency in C. elegans; (2) the AAUAAA is important in determining SL2 trans-splicing events more than 100 bp downstream; and (3) in some circumstances, 3′ end formation may occur by a trans-splicing-dependent mechanism.

Replication-dependent histone mRNAs end in a highly conserved 26-nt stem-loop structure. The stem-loop binding protein (SLBP), an evolutionarily conserved protein with no known homologs, interacts with the stem-loop in both the nucleus and cytoplasm and mediates nuclear-cytoplasmic transport as well as 3′-end processing of the pre-mRNA by the U7 snRNP. Here, we examined the affinity and specificity of the SLBP–RNA interaction. Nitrocellulose filter-binding experiments showed that the apparent equilibrium dissociation constant (Kd) between purified SLBP and the stem-loop RNA is 1.5 nM. Binding studies with a series of stem-loop variants demonstrated that conserved residues in the stem and loop, as well as the 5′ and 3′ flanking regions, are required for efficient protein recognition. Deletion analysis showed that 3 nt 5′ of the stem and 1 nt 3′ of the stem contribute to the binding energy. These data reveal that the high affinity complex between SLBP and the RNA involves sequence-specific contacts to the loop and the top of the stem, as well the base of the stem and its immediate flanking sequences. Together, these results suggest a novel mode of protein–RNA recognition that forms the core of a ribonucleoprotein complex central to the regulation of histone gene expression.

Ribonuclease P (RNase P) is the ribonucleoprotein enzyme that cleaves 5′-leader sequences from precursor-tRNAs. Bacterial and eukaryal RNase P RNAs differ fundamentally in that the former, but not the latter, are capable of catalyzing pre-tRNA maturation in vitro in the absence of proteins. An explanation of these functional differences will be assisted by a detailed comparison of bacterial and eukaryal RNase P RNA structures. However, the structures of eukaryal RNase P RNAs remain poorly characterized, compared to their bacterial and archaeal homologs. Hence, we have taken a phylogenetic-comparative approach to refine the secondary structures of eukaryal RNase P RNAs. To this end, 20 new RNase P RNA sequences have been determined from species of ascomycetous fungi representative of the genera Arxiozyma, Clavispora, Kluyveromyces, Pichia, Saccharomyces, Saccharomycopsis, Torulaspora, Wickerhamia, and Zygosaccharomyces. Phylogenetic-comparative analysis of these and other sequences refines previous eukaryal RNase P RNA secondary structure models. Patterns of sequence conservation and length variation refine the minimum-consensus model of the core eukaryal RNA structure. In comparison to bacterial RNase P RNAs, the eukaryal homologs lack RNA structural elements thought to be critical for both substrate binding and catalysis. Nonetheless, the eukaryal RNA retains the main features of the catalytic core of the bacterial RNase P. This indicates that the eukaryal RNA remains intrinsically a ribozyme.

Introns in plant nuclear pre-mRNAs are highly enriched in U or U + A residues and this property is essential for efficient splicing. Moreover, 3′-untranslated regions (3′-UTRs) in plant pre-mRNAs are generally UA-rich and contain sequences that are important for the polyadenylation reaction. Here, we characterize two structurally related RNA-binding proteins (RBPs) from Nicotiana plumbaginifolia, referred to as RBP45 and RBP47, having specificity for oligouridylates. Both proteins contain three RBD-type RNA-binding domains and a glutamine-rich N-terminus, and share similarity with Nam8p, a protein associated with U1 snRNP in the yeast Saccharomyces cerevisiae. Deletion analysis of RBP45 and RBP47 indicated that the presence of at least two RBD are required for interaction with RNA and that domains other than RBD do not significantly contribute to binding. mRNAs for RBP45 and RBP47 and mRNAs encoding six related proteins in Arabidopsis thaliana are constitutively expressed in different plant organs. Indirect immunofluorescence and fractionation of cell extracts showed that RBP45 and RBP47 are localized in the nucleus. In vivo UV crosslinking experiments demonstrated their association with the nuclear poly(A)+ RNA. In contrast to UBP1, another oligouridylate-binding nuclear three-RBD protein of N. plumbaginifolia (Lambermon et al., EMBO J, 2000, 19:1638–1649), RBP45 and RBP47 do not stimulate mRNA splicing and accumulation when transiently overexpressed in protoplasts. Properties of RBP45 and RBP47 suggest they represent hnRNP-proteins participating in still undefined steps of pre-mRNA maturation in plant cell nuclei.

U16 belongs to the family of box C/D small nucleolar RNAs (snoRNAs) whose members participate in ribosome biogenesis, mainly acting as guides for site-specific methylation of the pre-rRNA. Like all the other members of the family, U16 is associated with a set of protein factors forming a ribonucleoprotein particle, localized in the nucleolus. So far, only a few box C/D-specific proteins are known: in Xenopus laevis, fibrillarin and p68 have been identified by UV crosslinking and shown to require the conserved boxes C and D for snoRNA interaction. In this study, we have identified an additional protein factor (p62), common to box C/D snoRNPs, that crosslinks to the internal stem region, distinct from the conserved box C/D “core motif,” of U16 snoRNA. We show here that, although the absence of the core motif and, as a consequence, of fibrillarin and p68 binding prevents processing and accumulation of the snoRNA, the lack of the internal stem does not interfere with the efficient release of U16 from its host intron and only slightly affects snoRNA stability. Because this region is likely to be the binding site for p62, we propose that this protein plays an accessory role in the formation of a mature and stable U16 snoRNP particle.

A cell-free mRNA decay assay has been adapted to permit the kinetics of 3′ → 5′ exoribonuclease activities to be monitored in real time. RNA probes containing 5′ caps and 3′ poly(A) tails generated by transcription in vitro are 3′ labeled using fluorescein-N6-ATP and poly(A) polymerase. Release of fluorescein-conjugated adenosine residues from the 3′ end of the RNA substrate is monitored by a time-dependent decrease in fluorescence anisotropy in the presence of cytosolic proteins. To demonstrate the utility of the assay, an RNA probe was constructed containing a fragment of the c-myc 3′ untranslated region and an 85-base poly(A) tail. Following 3′ fluorescein labeling, the rate of 3′-terminal adenosine excision was monitored in the presence of an S100 cytosolic extract prepared from K562 erythroleukemia cells. Removal of the fluorescein-tagged A residues resolved to a first-order decay function, allowing the rate constant and enzyme-specific activity to be determined in this extract. Further applications and advantages of this technology are discussed.

Like its homologs in higher eukaryotes, the U2 snRNA in Schizosaccharomyces pombe is transcribed by RNA polymerase II and is not polyadenylated. Instead, an RNA stem-loop structure located downstream of the U2 snRNA coding sequence and transcribed as part of a 3′ extended precursor serves as a signal for 3′-end formation. We have identified three mutants that have temperature-sensitive defects in U2 snRNA 3′-end formation. In these mutants, the synthesis of the major snRNAs is also affected and unprocessed rRNA precursors accumulate at the restrictive temperature. Two of these mutants contain the same G-to-A transition within the pac1 gene, whereas the third contains a lesion outside the pac1 locus, indicating that at least two genes are involved. The pac1+ gene is codominant with the mutant allele and can rescue the temperature-sensitive phenotype and the defects in snRNA and rRNA synthesis, if overexpressed. In vitro, Pac1p, an RNase III homolog, can cleave a synthetic U2 precursor within the signal for 3′-end formation, generating a product that is a few nucleotides longer than mature U2 snRNA. In addition, U2 precursors are cleaved and trimmed to the mature size in extracts made from wild-type S. pombe cells. However, extracts made from pac1 mutant cells are unable to do so unless they are supplemented with purified recombinant Pac1p. Thus, the 3′ end of S. pombe U2 snRNA is generated by a processing reaction that requires Pac1p and an additional component, and can be dissociated from transcription in vitro.

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5′ external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3′ ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3′ extended form of the 25S rRNA. The 3′ extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3′ ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3′ ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3′ end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5′ end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3′ ETS is coupled.

In yeast, the 5′ end of the mature 18S rRNA is generated by endonucleolytic cleavage at site A1, the position of which is specified by two distinct signals. An evolutionarily conserved sequence immediately upstream of the cleavage site has previously been shown to constitute one of these signals. We report here that a conserved stem-loop structure within the 5′ region of the 18S rRNA is recognized as a second positioning signal. Mutations predicted to either extend or destabilize the stem inhibited the normal positioning of site A1 from within the 18S rRNA sequence, as did substitution of the loop nucleotides. In addition, these mutations destabilized the mature 18S rRNA, indicating that recognition of the stem-loop structure is also required for 18S rRNA stability. Several mutations tested reduced the efficiency of pre-rRNA cleavage at site A1. There was, however, a poor correlation between the effects of the different mutations on the efficiency of cleavage and on the choice of cleavage site, indicating that these involve recognition of the stem-loop region by distinct factors. In contrast, the cleavages at sites A1 and A2 are coupled and the positioning signals appear to be similar, suggesting that both cleavages may be carried out by the same endonuclease.

The rat β-tropomyosin (β-TM) gene encodes both skeletal muscle β-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1–5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3′ splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5′ splice site of exon 7 in nonmuscle cells. The 5′ splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5′ splice site of exon 7 does not result from the sequences at the 3′ end of intron 6 that block the use of the 3′ splice site of exon 7. However, mutating two conserved nucleotides GU at the 5′ splice site of exon 6 results in the efficient use of the 5′ splice site of exon 7. In addition, a mutation that changes the 5′ splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5′ splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells.

Ribosomal RNAs are generally synthesized as long, primary transcripts that must be extensively processed to generate the mature, functional species. In Escherichia coli, it is known that the initial 30S precursor is cleaved during its synthesis by the endonuclease RNase III to generate precursors to the 16S, 23S, and 5S rRNAs. However, despite extensive study, the processes by which these intermediate products are converted to their mature forms are poorly understood. In this article, we describe the maturation of 23S rRNA. Based on Northern analysis of RNA isolated from a variety of mutant strains lacking one or multiple ribonucleases, we show that maturation of the 3′ terminus requires the action of RNase T, an enzyme previously implicated in the end turnover of tRNA and in the maturation of small, stable RNAs. Although other exoribonucleases can participate in shortening the 3′ end of the initial RNase III cleavage product, RNase T is required for removal of the last few residues. In the absence of RNase T, 23S rRNA products with extra 3′ residues accumulate and are incorporated into ribosomes, with only small effects on cell growth. Purified RNase T accurately and efficiently converts these immature ribosomes to their mature forms in vitro, whereas free RNA is processed relatively poorly. In vivo, the processing defect at the 3′ end has no effect on 5′ maturation, indicating that the latter process proceeds independently. We also find that a portion of the 23S rRNA that accumulates in many RNase T− cells becomes polyadenylated because of the action of poly(A) polymerase I. The requirement for RNase T in 23S rRNA maturation is discussed in relation to a model in which only this enzyme, among the eight exoribonucleases present in E. coli, is able to efficiently remove nucleotides close to the double-stranded stem generated by the pairing of the 5′ and 3′ termini of most stable RNAs.

We have previously shown that a specific monoclonal antibody prepared against the U1A protein, MAb 12E12, is unique in its ability to recognize a form of U1A which is not associated with the U1snRNP. This unique form of U1A, termed snRNP-free U1A or SF-A, was found to be complexed with a novel set of non-snRNP proteins (O'Connor et al., 1997, RNA 3:1444-1455). Here we demonstrate that the largest protein in these SF-A complex(es), p105, is the polypyrimidine-tract binding protein-associated factor (PSF), an auxiliary splicing factor. We show that PSF copurifies and co-immunoprecipitates with SF-A from 293T cell nucleoplasm and that it interacts with SF-A in vitro. In addition, we show that MAb 12E12 inhibits both splicing and polyadenylation in an in vitro coupled splicing and polyadenylation reaction. This suggests that SF-A and/or the SF-A complex(es) perform an important function in both processing reactions and possibly in last exon definition.

A new category of self-splicing group I introns with conserved structural organization and function is found among the eukaryotic microorganisms Didymium and Naegleria. These complex rDNA introns contain two distinct ribozymes with different functions: a regular group I splicing-ribozyme and a small internal group I-like ribozyme (GIR1), probably involved in protein expression. GIR1 was found to cleave at two internal sites in an obligate sequential order. Both sites are located 3′ of the catalytic core. GIR1-catalyzed transesterification reactions could not be detected. We have compared all available GIR1 sequences and propose a common RNA secondary structure resembling that of group I splicing-ribozymes, but with some important differences. The GIR1s lack most peripheral sequence components, as well as a P1 segment, and, at approximately 160–190 nt, they are the smallest functional group I ribozymes known from nature. All GIR1s were found to contain a novel 6-bp pseudoknot (P15) within their catalytic core region. Experimental support of the proposed structure was obtained from the Didymium GIR1 by RNA structure probing and site-directed mutagenesis. Three-dimensional modeling indicates a compactly folded ribozyme with the functionally essential P15 exposed in the cleft between the two principal domains P3–P8 and P4–P6.