A 32-kilodalton protein binds to AU-rich domains in the 3'' untranslated regions of rapidly degraded mRNAs.

An AU-rich sequence present within the 3' untranslated region has been shown to mark some short-lived mRNAs for rapid degradation. We demonstrate by label transfer and gel shift experiments that a 32-kDa polypeptide, present in nuclear extracts, specifically interacts with the AU-rich domains present within the 3' untranslated region of human granulocyte-macrophage colony-stimulating factor, c-fos, and c-myc mRNAs and a similar domain downstream of the poly(A) addition site of the adenovirus IVa2 mRNA. Competition experiments and partial protease analysis indicated that the same polypeptide interacts with all four RNAs. A single AUUUA sequence in a U-rich context was sufficient to signal binding of the 32-kDa polypeptide. Insertion of three copies of this minimal recognition site led to markedly reduced accumulation of beta-globin RNA, while the same insert carrying a series of U-to-G changes had little effect on RNA levels. Steady-state levels of beta-globin-specific nuclear RNA, including incompletely processed RNA, and cytoplasmic mRNA were reduced. Cytoplasmic mRNA containing the AU-rich recognition sites for the 32-kDa polypeptide exhibited a half-life shorter than that of mRNA with a mutated insert. We suggest that binding of the 32-kDa polypeptide may be involved in the regulation of mRNA half-life.     

The 3'' untranslated regions of two related mRNAs contain an element highly repeated in the sea urchin genome.

Two closely related cDNA clones, pSpec1 and pSpec2, specifying two developmentally regulated tissue specific mRNAs from sea urchin embryos were used to probe a sea urchin genomic lambda library. Screening 10,000 phage by plaque hybridization yielded several hundred positive signals. With more stringent wash procedures, only two to three phage were positive. Three of these phage, one isolated by stringent wash procedures and two isolated by standard wash procedures were further investigated by restriction analysis, RNA gel blots, and DNA sequencing. The phage isolated by the stringent wash procedure appears to be a gene coding for the Specl mRNA. The other phage contain only partial homology to pSpec1 and pSpec2, 150 to 200 base pairs of the 3' untranslated region of the Spec1 and Spec2 mRNAs. It is concluded that the Spec1 and Spec2 mRNAs contain a highly repetitive element near their 3' end. The element is present at 2000 to 3000 copies per genome and may be transcribed at some sites other than those coding for the Spec1 and Spec2 genes. The possible function and evolutionary origin of the repetitive element is discussed.     

Expression of human alpha-tubulin genes: interspecies conservation of 3'' untranslated regions.

To examine the sequence complexity and differential expression of human alpha-tubulin genes, we constructed cDNA libraries from two unrelated tissue types (epidermis and fetal brain). The complete sequence of a positively hybridizing alpha-tubulin clone from each library is described. Each is shown to represent an abundantly expressed gene from fetal brain and keratinocytes, respectively. Although the coding regions are extensively homologous (97%), the 3' untranslated regions are totally dissimilar. This property has been used to dissect the human alpha-tubulin multigene family into members bearing sequence relatedness in this region. Surprisingly, each of these noncoding regions shares very high (65 to 80%) interspecies homology with the 3' untranslated region of one of the two rat alpha-tubulin genes of known sequence. These unexpected homologies imply the existence of selective pressure on the 3' untranslated regions of some cytoskeletal genes which maintains sequence fidelity during the course of evolution, perhaps as a consequence of an as yet unidentified functional requirement.     

UTRdb: a specialized database of 5' and 3' untranslated regions of eukaryotic mRNAs.

The 5' and 3' untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling mRNA localization, stability and translational efficiency. For this reason we developed UTRdb (http://bigarea.area.ba.cnr.it:8000/BioWWW/#U TRdb), a specialized database of 5' and 3' untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements.     

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.     

Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket

mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.     

Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs

The murine double minute 2 (mdm2) gene is essential for embryogenesis in mice that express the p53 tumor suppressor protein. Mdm2 levels must be regulated tightly because overexpression of mdm2 contributes to tumorigenesis. We investigated whether the 5' and 3' untranslated regions (UTRs) of murine mdm2 affect the expression of MDM2 proteins. Induction of mdm2 expression by p53 results in synthesis of an mdm2 mRNA with a short 5' UTR. The long 5' UTR increases internal initiation of translation of a minor MDM2 protein, p76(MDM2), without affecting the efficiency of translation of the full-length p90(MDM2). We discovered two alternative 3' untranslated regions in murine mdm2 mRNA expressed in the testis. The longer 3' UTR contains a consensus instability element, but mdm2 mRNAs containing the long and short 3' UTRs have comparable half-lives. The 3' UTRs do not affect either initiation codon use or translation efficiency. Thus, the murine 5' UTR, but not the 3'UTR, influences the ratio of the two MDM2 proteins but neither UTR affects MDM2 abundance significantly.     

The yeast nuclear gene CBS1 is required for translation of mitochondrial mRNAs bearing the cob 5'' untranslated leader

Summary Mitochondrial translation of the cob mRNA to yield apocytochrome b is specifically dependent on the nuclear gene CBS1, while mitochondrial translation of the oxi2 mRNA to yield cytochrome oxidase subunit III (cox III) is specifically dependent on the nuclear gene PET494. Chimeric oxi2 mRNAs bearing the 5 leaders of other mitochondrial mRNAs, transcribed from rho ^- mitochondrial DNAs termed MSU494, are translated in pet494 mutants. In this study, we examined translation of coxIII from MSU494-encoded chimeric mRNAs in zygotes of defined nuclear and mitochondrial genotype. CoxIII was translated from a chimeric mRNA bearing the cob leader only when the zygotes contained a wild-type CBS1 gene. CoxIII translation from an mRNA bearing the 5 leader of the mitochondrial gene aap1 was not dependent on CBS1 activity. We conclude that the product of the nuclear gene CBS1, or something under its control, acts in the mitochondrion on the cob mRNA 5 leader to activate translation of downstream coding sequences.     

Evidence for 3' untranslated region-dependent autoregulation of the Drosophila gene encoding the neuronal nuclear RNA-binding protein ELAV.

The Drosophila locus embryonic lethal abnormal visual system (elav) encodes a nuclear RNA-binding protein essential for normal neuronal differentiation and maintenance of neurons. ELAV is thought to play its role by binding to RNAs produced by other genes necessary for neuronal differentiation and consequently to affect their metabolism by an as yet unknown mechanism. ELAV structural homologues have been identified in a wide range of organisms, including humans, indicating an important conserved role for the protein. Analysis of elav germline transformants presented here shows that one copy of elav minigenes lacking a complete 3'' untranslated region (3'' UTR) rescues null mutations at elav, but that two copies are lethal. Additional in vivo experiments demonstrate that elav expression is regulated through the 3'' UTR of the gene and indicate that this level of regulation is dependent upon ELAV itself. Because ELAV is an RNA-binding protein, the simplest model to account for these findings is that ELAV binds to the 3'' UTR of its own RNA to autoregulate its expression. I discuss the implications of these results for normal elav function.     

The chicken dystrophin cDNA: striking conservation of the C-terminal coding and 3'' untranslated regions between man and chicken.

Dystrophin is a very large muscle protein (approximately 400 kd) the deficiency of which is responsible for Duchenne muscular dystrophy. Its function is unknown at present. In order to know whether different domains of the protein are differentially conserved during evolution, we have cloned and sequenced the chicken dystrophin cDNA. The protein coding sequence has almost the same size as in man. The N-terminal region that resembles the actin binding domain of alpha actinin, as well as the large spectrin like domain show 80% and 75% conservation respectively between chicken and man. In contrast, the C-terminal region shows 95% identity over 627 aa suggesting that it is an important region of interaction with other proteins. Comparison of the amino acid sequence of this C-terminal region to other protein sequences shows only marginally significant similarities. Finally we have found a striking conservation of three segments of the 3' untranslated sequence (85% homology over a total of 920 nt) between chicken and man. These also appear to be conserved in other mammals. This high conservation is not linked to open reading frames.