RNA splicing capability of live neuronal dendrites

Group II introns are commonly believed to be the progenitors of spliceosomal introns, but they are notably absent from nuclear genomes. Barriers to group II intron function in nuclear genomes therefore beg examination. A previous study showed that nuclear expression of a group II intron in yeast results in nonsensemediated decay and translational repression of mRNA, and that these roadblocks to expression are group II intron-specific. To determine the molecular basis for repression of gene expression, we investigated cellular dynamics of processed group II intron RNAs, from transcription to cellular localization. Our data show pre-mRNA mislocalization to the cytoplasm, where the RNAs are targeted to foci. Furthermore, tenacious mRNA-pre-mRNA interactions, based on intron-exon binding sequences, result in reduced abundance of spliced mRNAs. Nuclear retention of pre-mRNA prevents this interaction and relieves these expression blocks. In addition to providing a mechanistic rationale for group II intronspecific repression, our data support the hypothesis that RNA silencing of the host gene contributed to expulsion of group II introns from nuclear genomes and drove the evolution of spliceosomal introns.intron-mediated nuclear gene silencing | spliceosomal intron evolution G roup II introns that reside in genomes of bacteria, archaea, and eukaryotic organelles are ribozymes that self-splice from pre-mRNA transcripts independent of protein catalysis (1-3). Group II introns are also mobile retroelements that integrate into DNAs via an RNA intermediate (2, 3). Group II intron splicing is usually facilitated in vivo by an intron-encoded protein that acts as a maturase to help form the required secondary and tertiary structures (3). The intron RNA-protein complex is also required for group II intron retromobility. Both splicing and mobility of group II introns require interactions between exon-binding sequences (EBSs) within the intron and intron-binding sequences (IBSs) in the flanking exons of RNA or DNA targets (2, 3).The chemical steps of group II intron splicing are identical to those of nuclear spliceosomal introns (4, 5). There are also similarities of RNA sequences at the splice sites and of RNA structures within the ribozyme and the spliceosome (6-9). Because of these parallels, the catalytic group II introns are believed to be the progenitors of spliceosomal introns (6, 10, 11). It is widely speculated that group II introns entered the eukaryotic lineage with the mitochondrial endosymbiosis, invaded the nucleus, and evolved from RNA catalysts into efficient spliceosomedependent introns. However, group II introns are strikingly absent from modern nuclear genomes (1), which are replete with spliceosomal introns. It is still elusive how the ancestral group II introns might have evolved into spliceosomal introns or how they were expunged from nuclear genomes.As an initial effort to answer these questions, we had probed the fate of group II introns introduced into RNA polymerase II transcripts in Saccharomyces cerevis...

Section: Discussionmentioning

confidence: 99%

RNA–RNA interactions and pre-mRNA mislocalization as drivers of group II intron loss from nuclear genomes

Dong

Piazza

et al. 2014

“…Although the presence of a small proportion of Sm-class snRNPs in the cytoplasm is expected considering the cytoplasmic steps in their biogenesis, our data, together with previous studies by others, strongly argue against any significant level of U12-type splicing occurring outside the nucleus. Indeed, with the exception of some highly specialized cells (e.g., anucleate platelets) (37) or in the case of neuronal dendrites (38), there is little evidence that pre-mRNA splicing in general takes place outside of the nucleus. Furthermore, the splicing of both U2-and U12-type introns is routinely performed in vitro in nuclear extract, and, in the absence of added factors (i.e., SR proteins), neither spliceosome is active in cytoplasmic extracts (39).…”

Section: Discussionmentioning

confidence: 99%

Minor spliceosome components are predominantly localized in the nucleus

Pessa

Will

Meng³

et al. 2008

Recently, it has been reported that there is a differential subcellular distribution of components of the minor U12-dependent and major U2-dependent spliceosome, and further that the minor spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and minor spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12-and U2-dependent spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major spliceosome, components of the U12-dependent spliceosome are localized predominantly in the nucleus.localization ͉ pre-mRNA splicing ͉ snRNA ͉ U12-dependent spliceosome P re-mRNA splicing is an essential step in the co/posttranscriptional processing of eukaryotic transcripts before their export from the nucleus. The low-abundance U12-dependent ''minor'' spliceosome exists in parallel with the U2-dependent ''major'' spliceosome in most multicellular eukaryotes (1). It catalyzes the removal of a rare class of introns (U12-type) that represent Ͻ1% of introns in mammals (2, 3). The U12-dependent spliceosome contains four unique snRNAs: U11, U12, U4atac, and U6atac, which are paralogs of U1, U2, U4, and U6 snRNAs of the U2-dependent spliceosome, respectively (4, 5). U5 snRNA, in contrast, is shared between the two spliceosomes. In addition to the snRNAs, both spliceosomes contain numerous snRNP and non-snRNP proteins (6, 7).The five snRNP components of the major spliceosome are predominantly localized in the nucleus, but the biogenesis of four of them involves a cytoplasmic step (8). That is, after transcription by RNA Pol II, the snRNAs U1, U2, U4, and U5, which all contain an Sm protein-binding site, are first exported into the cytoplasm. The Sm proteins subsequently bind, and the snRNA's cap is hypermethylated to 2,2,7-tri-methyl-guanosine (m 3 G) (9, 10). These processing steps are a prerequisite for their reimport into the nucleus (11). In contrast to these so-called Sm-class snRNAs, U6 snRNA is transcribed by RNA pol III, acquires Sm-like proteins (Lsm2-8) (12) and a ␥-monomethyl phosphate cap, and does not leave the nucleus. The minor snRNAs U11, U12, and U4atac are also Sm-class snRNAs, containing an Sm-binding site that is also bound by Sm proteins and an m 3 G cap (5, 13), whereas U6atac is transcribed by RNA pol III, possesses a ␥-monomethyl phosphate cap (5), and is assembled with Lsm proteins (14). After nuclear import, spliceosomal snRNPs first pass through Cajal bodies, where they undergo further modifications and assembly (15, 16) and then subsequentl...

“…It might be postulated that long premRNAs are reimported to the nucleus for further processing when the cell needs to generate monocistrons for translation. However, it has recently been described that splicing might occur in the cytosol of some types of anucleate cells and in neuronal dendrites having a functional cytosolic spliceosome complex (35,36).…”

Section: Discussionmentioning

confidence: 99%

mRNA maturation by two-step trans-splicing/polyadenylation processing in trypanosomes

Jäger

Gaudenzi

Cassola

et al. 2007

Trypanosomes are unique eukaryotic cells, in that they virtually lack mechanisms to control gene expression at the transcriptional level. These microorganisms mostly control protein synthesis by posttranscriptional regulation processes, like mRNA stabilization and degradation. Transcription in these cells is polycistronic. Tens to hundreds of protein-coding genes of unrelated function are arrayed in long clusters on the same DNA strand. Polycistrons are cotranscriptionally processed by trans-splicing at the 5 end and polyadenylation at the 3 end, generating monocistronic units ready for degradation or translation. In this work, we show that some trans-splicing/polyadenylation sites may be skipped during normal polycistronic processing. As a consequence, dicistronic units or monocistronic transcripts having long 3 UTRs are produced. Interestingly, these unspliced transcripts can be processed into mature mRNAs by the conventional trans-splicing/ polyadenylation events leading to translation. To our knowledge, this is a previously undescribed mRNA maturation by trans-splicing uncoupled from transcription. We identified an RNA-recognition motif-type protein, homologous to the mammalian polypyrimidine tract-binding protein, interacting with one of the partially processed RNAs analyzed here that might be involved in exon skipping. We propose that splice-site skipping might be part of a posttranscriptional mechanism to regulate gene expression in trypanosomes, through the generation of premature nontranslatable RNA molecules.gene expression ͉ posttranscriptional regulation ͉ RNA processing R egulation of gene expression is central in defining the phenotype of a cell or organism. In the vast majority of cases, this regulation is controlled by transcriptional regulation in which DNA sequence elements, together with DNA-binding proteins, target genes for transcription by RNA polymerase II. In addition to regulating single genes, master regulators of transcription may give rise to a gene expression phenotype, a trait that may be inherited (1). Mature transcripts can also be regulated in their expression at the posttranscriptional level. A number of cis-motifs mainly located in the 5Ј and 3Ј UTRs determine the fate of the transcript by the use of highly specific and sometimes evolutionarily conserved machineries. This process is essentially achieved through RNA-binding proteins (RBPs) forming, together with the mRNA, ribonucleoprotein (RNP) complexes. It is the composition of the RNP complex that determines whether an mRNA is transported to the cytoplasm for translation, degradation, or other processing events (2, 3).Posttranscriptional regulation is common among Kinetoplastid parasites, like trypanosomes and Leishmania (4, 5), which are the causative agents of several infections affecting both humans and domestic animals worldwide (6). In these parasites, transcription by RNA polymerase II starts at a few genomic locations within chromosomes. In contrast to operons in bacteria, polycistronic units in trypanosomatids requir...