Other Drosophila hnRNPs

The Drosophila melanogaster hnRNP protein, hrp48, is an abundant heterogeneous nuclear RNA-associated protein. Previous biochemical studies have implicated hrp48 as a component of a ribonucleoprotein complex involved in the regulation of the tissue-specific alternative splicing of the P-element third intron (IVS3). A genetic approach was taken to analyzing the role of hrp48. Mutations in the hrp48 gene have been identified and characterized. hrp48 is an essential gene. Hypomorphic mutations which reduce the level of hrp48 protein display developmental defects, including reduced numbers of ommatidia in the eye and morphological bristle abnormalities. Using a P-element third-intron reporter transgene, reduced levels of hrp48 were found to partially relieve IVS3 splicing inhibition in somatic cells. This is the first direct evidence that hrp48 plays a functional role in IVS3 splicing inhibition (Hammond, 1997).

Alternatively spliced Ultrabithorax mRNAs differ by the presence of internal exons mI and mII. Two approaches were used to identify trans-acting factors required for inclusion of these cassette exons. First, mutations in a set of genes implicated in the control of other alternative splicing decisions were tested for dominant effects on the Ubx alternative splicing pattern. To identify additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested first for dominant enhancement of the haploinsufficient Ubx haltere phenotype and second for effects on the splicing pattern. Inclusion of the cassette exons in Ubx mRNAs Is reduced strongly in heterozygotes for hypomorphic alleles of hrp48, which encodes a member of the hnRNP A/B family and is implicated in control of P-element splicing. Significant reductions of mI and mII inclusion are also observed in heterozygotes for loss-of-function alleles of virilizer, fl(2)d, and crooked neck. The products of virilizer and fl(2)d are also required for Sxl autoregulation at the level of splicing; crooked neck encodes a protein with structural similarities to yeast-splicing factors Prp39p and Prp42p. Deletion of at least five other loci caused significant reductions in the inclusion of mI and/or mII. Possible roles of identified factors are discussed in the context of the resplicing strategy for generation of alternative Ubx mRNAs (Burnette, 1999).

Cloning and alternative splicing of hnRNPs

The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. Two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bind specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. The RNA-binding component of this mRNA degradation activity, which is referred to as AUF1 has been purified. A+U-rich (ARE) binding/degradation factor (AUF1) family of proteins are also known as the heterogeneous nuclear RNP (hnRNP) D proteins. Using antisera specific for this protein, it is demonstrated that the 37- and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation (Zhang, 1993).

The hnRNP D protein interacts with nucleic acids both in vivo and in vitro. Like many other proteins that interact with RNA, it contains RBD (or "RRM") domains and arg-gly-gly (RGG) motifs. The organization and localization were examined of the human and murine genes that encode the hnRNP D protein. Comparison of the predicted sequences of the hnRNP D proteins in human and mouse shows that they are 96.9% identical (98.9% similar). This very high level of conservation suggests a critical function for hnRNP D. Sequence analysis of the human HNRPD gene shows that the protein is encoded by eight exons and that two additional exons specify sequences in the 3' UTR. Use of two of the coding exons is determined by alternative splicing of the HNRPD mRNA. The human HNRPD gene maps to 4q21. The mouse Hnrpd gene maps to the F region of chromosome 3, which is syntenic with the human 4q21 region (Dempsey, 1998a).

The human DNA- and RNA-binding protein JKTBP is a member of a 2xRNA-binding domain (RBD)-glycine family of heterogeneous nuclear ribonucleoproteins that are involved in mRNA biogenesis. Northern and Western blottings reveal that mRNAs of approx. 1.4 and 2.8kb and proteins of approx. 38 and 53kDa are present in HL-60 cells and various tissues. Cloning and characterization of a previously unknown cDNA for the 2.8kb mRNA indicates that the cDNA encodes a 420 amino acid JKTBP polypeptide. Isolation and characterization of the genomic DNA shows that the gene (HNRPDL) has nine exons and has two separate transcription start sites for the two transcripts. The features of the 5' flanking sequences of these sites shows that the gene is a housekeeping gene. Fluorescence in situ hybridization mapped the gene to 4q13-q21. From its gene organization, the JKTBP seems to be most closely related to hnRNP D/AUF1 (Kamei, 1999).

Several proteins that may regulate c-myc mRNA post-transcriptionally have been isolated and characterized. Two of them, HuR and AUF1, bind specifically to the 3' untranslated region (UTR) of c-myc mRNA. Because c-myc is regulated post-transcriptionally in various mouse tissues, including quiescent tissues, fetal liver and regenerating liver, an investigation was carried out to see whether HuR and AUF1. expression is also regulated in these tissues. Concerning AUF1, the expression of various mRNA and protein isoforms were examined. A new AUF1 mRNA variant has been discoved with a long AU-rich 3' UTR. AUF1 expression, regardless of the RNA isoform considered, and HuR mRNA expression parallel c-myc expression in quiescent tissues and during liver development; their expression is high in lymphoid tissues and fetal liver and low in adult liver. However, no upregulation of HuR or AUF1 accompanies the upregulation of c-myc mRNA following partial hepatectomy. These results are discussed in relation to the current hypothesis that HuR and AUF1 act as mRNA destabilizing factors (Lafon, 1998).

The steady-state levels of many mRNAs are determined in part by their turnover rates. Turnover rates, in turn, are usually controlled by proteins that bind cis-acting sequence elements in mRNAs. One class of cis-acting instability determinants is composed of A + U-rich elements present in the 3'-UTRs of many labile mRNAs. Many A + U-rich elements are bound by the AUF1 family of RNA-binding proteins, which may target these mRNAs for rapid decay. cDNA cloning and immunoblot analyses suggest that the AUF1 family consists of at least four isoforms. Previous genomic cloning combined with FISH and Southern analyses of a panel of monochromosomal mouse/human or hamster/human somatic cell hybrids localized two AUF1 loci to human 4q21.1-q21.2 and Xq12. In the present study AUF1 gene organization was examined. The results suggest that the four known AUF1 isoforms are generated by alternative pre-mRNA splicing of a transcript encoded by the chromosome 4 locus. Functionally, this creates isoforms with different RNA-binding affinities and specificities. Thus, alternative pre-mRNA splicing may serve to create functional versatility within the AUF1 family of proteins (Wagner, 1998).

The A+U-rich RNA-binding factor AUF1 exhibits characteristics of a trans-acting factor contributing to the rapid turnover of many cellular mRNAs. Structural mapping of the AUF1 gene and its transcribed mRNA has revealed alternative splicing events within the 3' untranslated region (3'-UTR). In K562 erythroleukemia cells, four alternatively spliced AUF1 3'-UTR variants have been identified, including a population of AUF1 mRNA containing a highly conserved 107-nucleotide (nt) 3'-UTR exon (exon 9) and the adjacent downstream intron (intron 9). Functional analyses using luciferase-AUF1 3'-UTR chimeric transcripts demonstrates that the presence of either a spliceable or an unspliceable intron 9 in the 3'-UTR represses luciferase expression in cis, indicating that intron 9 sequences may down-regulate gene expression by two distinct mechanisms. In the case of the unspliceable intron, repression of luciferase expression likely involved two AUF1-binding sequences, since luciferase expression was increased by deletion of these sites. However, inclusion of the spliceable intron in the luciferase 3'-UTR down-regulates expression independent of the AUF1-binding sequences. This is likely due to nonsense-mediated mRNA decay (NMD) owing to the generation of exon-exon junctions more than 50 nt downstream of the luciferase termination codon. AUF1 mRNA splice variants generated by selective excision of intron 9 are thus also likely to be subject to NMD since intron 9 is always positioned >137 nt downstream of the stop codon. The distribution of alternatively spliced AUF1 transcripts in K562 cells is consistent with this model of regulated AUF1 expression (Wilson, 1999).

Identification of mRNAs bound by hnRNPs

Cell activation is associated with diverse and widespread changes in gene expression at both the transcriptional and post-transcriptional levels. AUF1 is a recently described cytoplasmic protein which likely participates in the post-transcriptional regulation (PTR) of AU-rich (ARE) mRNAs including those coding for cytokines and proto-oncogenes. Individual mRNAs subject to AUF1-mediated PTR can be predicted if AREs are present or the mRNA in question interacts in vitro or in vivo with AUF1. However, there are few, if any, general approaches for characterizing the overall repertoire of mRNAs subject to PTR by AUF1. In an effort to identify these mRNAs, total mRNA from mitogen-activated peripheral blood mono-nuclear cells (PBMCs) was incubated with AUF1 in vitro. AUF1-mRNA complexes were retarded on membranes, bound mRNAs eluted with high salt, and either used to generate a cDNA library or rebound to AUF1 a second or third time prior to elution and cDNA library construction. Partial nucleotide sequences were obtained from 130 clones which shows that the AUF1 selected libraries are rich in mRNAs containing 3' untranslated region AREs including a large number of early response gene cDNAs. As a test of the validity of this method, a randomly selected, novel mRNA contained in the library is shown to be stabilized upon cell activation (Bhattacharya, 1999).

The expression of CPEB proteins is sequentially regulated during zebrafish oogenesis and embryogenesis

In many species there is little transcription in the mature oocyte, and zygotic transcription does not begin immediately after fertilization; this is the case in zebrafish, where zygotic transcription is not initiated until the mid-blastula transition. Thus the production of new proteins during oogenesis and early embryogenesis is dependent on the translation of maternal mRNAs. In a growing number of species, the translation of key maternal transcripts is coupled to their cytoplasmic polyadenylation. One family of RNA-binding proteins implicated in this process are the cytoplasmic polyadenylation element (CPE)-binding proteins (CPEBs), which bind to a sequence in the 3'- untranslated regions (3'-UTRs) of regulated transcripts and mediate their storage/repression or translation. In several species, there is evidence for two classes of CPEBs, a larger oocyte-type and a smaller CPEB that functions during embryogenesis. This appears to be the case in zebrafish as well, and this study provides evidence suggesting that the oocyte-type CPEB (zorba) regulates the translation of the embryonic-type (ElrA) by keeping the ElrA transcript in storage until fertilization. When zorba levels fall, the ElrA protein is then produced and available to regulate the translation of additional mRNAs during embryogenesis. A potential target of ElrA, the maternal mRNA for hnRNPab, was identified that is a potential homolog of the Drosophila gene squid, whose product plays a role in patterning the Drosophila oocyte and embryo. These data suggest that during zebrafish embryogenesis, cytoplasmic polyadenylation mediates a cascade of translational control whose final targets play central patterning roles during embryogenesis (O'Connell, 2014).

Domain and three dimensional structure of hnRNPs

AUF1 is an RNA-binding protein that contains two nonidentical RNA recognition motifs (RRMs). AUF1 binds to A + U-rich elements (AREs) with high affinity. The binding of AUF1 to AREs is believed to serve as a signal to an mRNA-processing pathway that degrades mRNAs encoding many cytokines, oncoproteins, and G protein-coupled receptors. Because the ARE binding activity of AUF1 appears central to the regulation of many important genes, the domains of the protein that are important for this activity were examined. Examination of the RNA binding affinity of various AUF1 mutants suggests that both RRMs may be required for binding to the human c-fos ARE. However, the two RRMs together are not sufficient. Highest affinity binding of AUF1 to an ARE requires an alanine-rich region of the N terminus and a short glutamine-rich region in the C terminus. In addition, the N terminus is required for dimerization of AUF1. However, AUF1 binds an ARE as a hexameric protein. Thus, protein-protein interactions are important for high affinity ARE binding activity of AUF1 (DeMaria, 1997).

Human hnRNP A1 is a versatile single-stranded nucleic acid-binding protein that functions in various aspects of mRNA maturation and in telomere length regulation. The crystal structure of UP1, the amino-terminal domain of human hnRNP A1 containing two RNA-recognition motifs (RRMs), bound to a 12-nucleotide single-stranded telomeric DNA has been determined at 2.1 A resolution. The structure of the complex reveals the basis for sequence-specific recognition of the single-stranded overhangs of human telomeres by hnRNP A1. It also provides insights into the basis for high-affinity binding of hnRNP A1 to certain RNA sequences, and for nucleic acid binding and functional synergy between the RRMs. In the crystal structure, a UP1 dimer binds to two strands of DNA, and each strand contacts RRM1 of one monomer and RRM2 of the other. The two DNA strands are antiparallel, and regions of the protein flanking each RRM make important contacts with DNA. The extensive protein-protein interface seen in the crystal structure of the protein-DNA complex and the evolutionary conservation of the interface residues suggest the importance of specific protein-protein interactions for the sequence-specific recognition of single-stranded nucleic acids. Models for regular packaging of telomere 3' overhangs and for juxtaposition of alternative 5' splice sites are proposed (Ding, 1999).

Involvement of hnRNPs in mRNA assembly and splicing

In the active Balbiani ring (BR) genes of the dipteran Chironomus tentans, the assembly of a specific pre-mRNP particle can be analyzed in situ, and the incorporation of hnRNP proteins into the nascent pre-mRNP can be directly visualized by immunoelectron microscopy. hrp36, one of the major hnRNP proteins in Chironomus tentans, is continuously added to the nascent BR pre-mRNP particle throughout transcription and is localized along the entire BR RNP fiber. Interestingly, hrp36 becomes concealed during the structural transition that occurs during the formation of the mature BR RNP particle. This conclusion is based on the observation that hrp36 can be revealed by a monoclonal antibody during the initial assembly of the BR RNP fiber but becomes almost undetectable in the final packaging stage. The hrp36 protein, however, is not removed from the BR RNP particle since the ability of the monoclonal antibody to reveal hrp36 is restored by artificial relaxation of mature BR RNP particles. Another major hnRNP protein, hrp45, is also incorporated in a continuous manner into the nascent pre-mRNP fiber but remains accessible in mature BR RNP particles. These results provide immunocytochemical evidence for drastic structural changes occurring in the final stage of BR pre-mRNP packaging, and suggest that different hnRNP proteins might be differently involved in the pre-mRNP assembly process (Kiseleva, 1997).

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, it is proposed that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. An intron element was also identified which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. These results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing (Blanchette, 1999).

Splicing of the human immunodeficiency virus type 1 (HIV-1) pre-mRNA must be inefficient to provide a pool of unspliced messages which encode viral proteins and serve as genomes for new virions. Negative cis-regulatory elements (exonic splicing silencers or ESSs) are necessary for HIV-1 splicing inhibition. Heterogeneous nuclear ribonucleoproteins (hnRNPs) of the A and B group are trans-acting factors required for the function of the tat exon 2 ESS. Depletion of hnRNP A/B proteins from HeLa cell nuclear extract activates splicing of tat exon 2 pre-mRNA substrate. Splicing inhibition is restored by addition of recombinant hnRNP A/B proteins to the depleted extract. A high-affinity hnRNP A1-binding sequence can substitute functionally for the ESS in tat exon 2. These results demonstrate that hnRNP A/B proteins are required for repression of HIV-1 splicing (Capeti, 1999).

squid Evolutionary homologs part 2/2

squid: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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