Sonic hedgehog expression in zebrafish forebrain identifies the teleostean pallidal signaling center and shows preglomerular complex and posterior tubercular dopamine cells to arise from shh cells

Ventralization, a major patterning process in the developing vertebrate neural tube (central nervous system, CNS), depends on Sonic hedgehog (SHH) as a main signaling morphogen. We studied the CNS of late larval and young adult zebrafish in a transgenic shh‐GFP line revealing increased neuroanatomical detail due to the progressed differentiation state compared to earlier stages. Some major findings emerge from the present study. (a) shh –GFP is still expressed along the adult zebrafish CNS neuraxis in most locations seen in larvae. (b) We newly identify a ventroposterior shh pallidal domain representing the basal telencephalic signaling center important for basal ganglia development known in other vertebrates (i.e., the anterior entopeduncular area—basal medial ganglionic eminence of mammals). (c) We further show late‐emerging shh‐GFP positive radial glia cells in the medial zone of the dorsal telencephalon (i.e., the teleostan pallial amygdala). (d) Immunostains for tyrosine hydroxylase demonstrate that there is selective colocalization in adult dopamine cells with shh‐GFP in the posterior tuberculum, including in projection cells to striatum, which represents a striking parallel to amniote mesodiencephalic dopamine cell origin from shh expressing floor plate cells. (e) There is no colocalization of shh and islet1 as shown by respective shh‐GFP and islet1‐GFP lines. (f) The only radially far migrated shh‐GFP cells are located in the preglomerular area. (g) There are no adult cerebellar and tectal shh‐GFP cells confirming their exclusive role during early development as previously reported by our laboratory

Gene processing control loops suggested by sequencing, splicing, and RNA folding

Abstract Background Small RNAs are known to regulate diverse gene expression processes including translation, transcription, and splicing. Among small RNAs, the microRNAs (miRNAs) of 17 to 27 nucleotides (nts) undergo biogeneses including primary transcription, RNA excision and folding, nuclear export, cytoplasmic processing, and then bioactivity as regulatory agents. We propose that analogous hairpins from RNA molecules that function as part of the spliceosome might also be the source of small, regulatory RNAs (somewhat smaller than miRNAs). Results Deep sequencing technology has enabled discovery of a novel 16-nt RNA sequence in total RNA from human brain that we propose is derived from RNU1, an RNA component of spliceosome assembly. Bioinformatic alignments compel inquiring whether the novel 16-nt sequence or its precursor have a regulatory function as well as determining aspects of how processing intersects with the miRNA biogenesis pathway. Specifically, our preliminary in silico investigations reveal the sequence could regulate splicing factor Arg/Ser rich 1 (SFRS1), a gene coding an essential protein component of the spliceosome. All 16-base source sequences in the UCSC Human Genome Browser are within the 14 instances of RNU1 genes listed in wgEncodeGencodeAutoV3. Furthermore, 10 of the 14 instances of the sequence are also within a common 28-nt hairpin-forming subsequence of RNU1. Conclusions An abundant 16-nt RNA sequence is sourced from a spliceosomal RNA, lies in a stem of a predicted RNA hairpin, and includes reverse complements of subsequences of the 3'UTR of a gene coding for a spliceosome protein. Thus RNU1 could function both as a component of spliceosome assembly and as inhibitor of production of the essential, spliceosome protein coded by SFRS1. Beyond this example, a general procedure is needed for systematic discovery of multiple alignments of sequencing, splicing, and RNA folding data

Differentiating Protein-Coding and Noncoding RNA: Challenges and Ambiguities

The assumption that RNA can be readily classified into either protein-coding or non-protein–coding categories has pervaded biology for close to 50 years. Until recently, discrimination between these two categories was relatively straightforward: most transcripts were clearly identifiable as protein-coding messenger RNAs (mRNAs), and readily distinguished from the small number of well-characterized non-protein–coding RNAs (ncRNAs), such as transfer, ribosomal, and spliceosomal RNAs. Recent genome-wide studies have revealed the existence of thousands of noncoding transcripts, whose function and significance are unclear. The discovery of this hidden transcriptome and the implicit challenge it presents to our understanding of the expression and regulation of genetic information has made the need to distinguish between mRNAs and ncRNAs both more pressing and more complicated. In this Review, we consider the diverse strategies employed to discriminate between protein-coding and noncoding transcripts and the fundamental difficulties that are inherent in what may superficially appear to be a simple problem. Misannotations can also run in both directions: some ncRNAs may actually encode peptides, and some of those currently thought to do so may not. Moreover, recent studies have shown that some RNAs can function both as mRNAs and intrinsically as functional ncRNAs, which may be a relatively widespread phenomenon. We conclude that it is difficult to annotate an RNA unequivocally as protein-coding or noncoding, with overlapping protein-coding and noncoding transcripts further confounding this distinction. In addition, the finding that some transcripts can function both intrinsically at the RNA level and to encode proteins suggests a false dichotomy between mRNAs and ncRNAs. Therefore, the functionality of any transcript at the RNA level should not be discounted

Analyses of In Vivo Interaction and Mobility of Two Spliceosomal Proteins Using FRAP and BiFC

U1-70K, a U1 snRNP-specific protein, and serine/arginine-rich (SR) proteins are components of the spliceosome and play critical roles in both constitutive and alternative pre-mRNA splicing. However, the mobility properties of U1-70K, its in vivo interaction with SR proteins, and the mobility of the U1-70K-SR protein complex have not been studied in any system. Here, we studied the in vivo interaction of U1-70K with an SR protein (SR45) and the mobility of the U1-70K/SR protein complex using bimolecular fluorescence complementation (BiFC) and fluorescence recovery after photobleaching (FRAP). Our results show that U1-70K exchanges between speckles and the nucleoplasmic pool very rapidly and that this exchange is sensitive to ongoing transcription and phosphorylation. BiFC analyses showed that U1-70K and SR45 interacted primarily in speckles and that this interaction is mediated by the RS1 or RS2 domain of SR45. FRAP analyses showed considerably slower recovery of the SR45/U1-70K complex than either protein alone indicating that SR45/U1-70K complexes remain in the speckles for a longer duration. Furthermore, FRAP analyses with SR45/U1-70K complex in the presence of inhibitors of phosphorylation did not reveal any significant change compared to control cells, suggesting that the mobility of the complex is not affected by the status of protein phosphorylation. These results indicate that U1-70K, like SR splicing factors, moves rapidly in the nucleus ensuring its availability at various sites of splicing. Furthermore, although it appears that U1-70K moves by diffusion its mobility is regulated by phosphorylation and transcription

Cloning of a gene (SR-A1), encoding for a new member of the human Ser/Arg-rich family of pre-mRNA splicing factors: overexpression in aggressive ovarian cancer

By using the positional cloning gene approach, we were able to identify a novel gene encoding for a serine/arginine-rich protein, which appears to be the human homologue of the rat A1 gene. We named this new gene SR-A1. Members of the SR family of proteins have been shown to interact with the C-terminal domain (CTD) of the large subunit of RNA polymerase II and participate in pre-mRNA splicing. We have localized the SR-A1 gene between the known genes IRF3 and RRAS on chromosome 19q13.3. The novel gene spans 16.7 kb of genomic sequence and it is formed of 11 exons and 10 intervening introns. The SR-A1 protein is composed of 1312 amino acids, with a molecular mass of 139.3 kDa and a theoretical isoelectric point of 9.31. The SR-A1 protein contains an SR-rich domain as well as a CTD-binding domain present only in a subset of SR-proteins. Through interactions with the pre-mRNA and the CTD domain of the Polymerase II, SR proteins have been shown to regulate alternative splicing. The SR-A1 gene is expressed in all tissues tested, with highest levels found in fetal brain and fetal liver. Our data suggest that this gene is overexpressed in a subset of ovarian cancers which are clinically more aggressive. Studies with the steroid hormone receptor-positive breast and prostate carcinoma cell lines ZR-75-1, BT-474 and LNCaP, respectively, suggest that SR-A1 is constitutively expressed. Furthermore, the mRNA of the SR-A1 gene in these cell lines appears to increase by estrogens, androgens and glucocorticoids, and to a lesser extend by progestins. © 2001 Cancer Research Campaign http://www.bjcancer.co

Deletion of the N-terminus of SF2/ASF Permits RS-Domain-Independent Pre-mRNA Splicing

Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing

Regulation of Plant Developmental Processes by a Novel Splicing Factor

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes

Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

The shape of nuclei in many adherent cultured cells approximates an oblate ellipsoid, with contralateral flattened surfaces facing the culture plate or the medium. Observations of cultured cell nuclei from orthogonal perspectives revealed that nucleoporin p62 (NUP62) and nucleoporin 214 (NUP214) are differentially distributed between nuclear pore complexes on the flattened surfaces and peripheral rim of the nucleus. High resolution stimulated emission depletion (STED) immunofluorescence microscopy resolved individual NPCs, and suggested both heterogeneity and microheterogeneity in NUP62 and NUP214 immunolabeling among in NPC populations. Similar to nuclear domains and interphase chromosome territories, architectural diversity and spatial patterning of NPCs may be an intrinsic property of the nucleus that is linked to the functions and organization of underlying chromatin

The Genetic Signatures of Noncoding RNAs

The majority of the genome in animals and plants is transcribed in a developmentally regulated manner to produce large numbers of non–protein-coding RNAs (ncRNAs), whose incidence increases with developmental complexity. There is growing evidence that these transcripts are functional, particularly in the regulation of epigenetic processes, leading to the suggestion that they compose a hitherto hidden layer of genomic programming in humans and other complex organisms. However, to date, very few have been identified in genetic screens. Here I show that this is explicable by an historic emphasis, both phenotypically and technically, on mutations in protein-coding sequences, and by presumptions about the nature of regulatory mutations. Most variations in regulatory sequences produce relatively subtle phenotypic changes, in contrast to mutations in protein-coding sequences that frequently cause catastrophic component failure. Until recently, most mapping projects have focused on protein-coding sequences, and the limited number of identified regulatory mutations have been interpreted as affecting conventional cis-acting promoter and enhancer elements, although these regions are often themselves transcribed. Moreover, ncRNA-directed regulatory circuits underpin most, if not all, complex genetic phenomena in eukaryotes, including RNA interference-related processes such as transcriptional and post-transcriptional gene silencing, position effect variegation, hybrid dysgenesis, chromosome dosage compensation, parental imprinting and allelic exclusion, paramutation, and possibly transvection and transinduction. The next frontier is the identification and functional characterization of the myriad sequence variations that influence quantitative traits, disease susceptibility, and other complex characteristics, which are being shown by genome-wide association studies to lie mostly in noncoding, presumably regulatory, regions. There is every possibility that many of these variations will alter the interactions between regulatory RNAs and their targets, a prospect that should be borne in mind in future functional analyses