Multiple roles of the coding sequence 5′ end in gene expression regulation

The codon composition of the coding sequence''s (ORF) 5′ end first few dozen codons is known to be distinct to that of the rest of the ORF. Various explanations for the unusual codon distribution in this region have been proposed in recent years, and include, among others, novel regulatory mechanisms of translation initiation and elongation. However, due to the fact that many overlapping regulatory signals are suggested to be associated with this relatively short region, its research is challenging. Here, we review the currently known signals that appear in this region, the theories related to the way they regulate translation and affect the organismal fitness, and the debates they provoke.     

Effect of deletions 5′ to the translation initiation sequence on the expression of an mRNA in animal cells

To learn if an mRNA·18S rRNA interaction or a special secondary structure in the mRNA start region is essential for translation in eukaryotic cells, we constructed recombinant plasmids with the SV40 early promoter 5 to part of the Escherichia coli tuf B-lacZ gene. Deletion of bases potentially complementary to the 18S rRNA highly increased the transient -galactosidase expressed in transfected CHO cells. Deletion of bases that fostered formation of potential hairpins with the mRNA 5-terminus or altered the structure of the coding region reduced -galactosidase activity suggesting that these features of the mRNA secondary structure may be essential for initiation of translation. Computer aided analysis of the potential structure of 290 mRNAs suggests these are conserved features of the initiation region.     

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.     

5′ Flanking sequence of the hamster desmin gene

Molecular Biology Reports -     

Unusual sequence conservation in the 5′ and 3′ untranslated regions of the sea urchin spec mRNAs

Summary The Spec1 and Spec2 mRNAs (Strongylocentrotus purpuratus ectoderm mRNAs) represent a small gene family that encodes 10–12 members of the troponin C superfamily of calcium-binding proteins. These mRNAs and proteins accumulate in the aboral (dorsal) ectoderm of sea urchin embryos and larvae. Using genomic and cDNA clones, we have compared the sequences of four Spec mRNAs: Spec1, Spec2a, Spec2c, and Spec2d. The mRNAs all have at least 120 bases of 5 untranslated leader, approximately 450 bases of open reading frame, and 900 bases (Spec1) or 1250 bases (Spec2a, 2c, 2d) of 3 untranslated trailer. Unexpectedly, when long stretches of 5 untranslated regions or 3 untranslated regions are compared to one another, they are found to be less divergent than the protein-coding regions. Comparing Spec2d, the most divergent member of the family, with the other Spec mRNAs shows that while the protein-coding regions are 60–62% matched, the untranslated regions are greater than 80% matched. Comparisons among Spec1, Spec2a, and Spec2c demonstrate similar but less dramatic conservation of untranslated regions. Our data imply that the Spec gene family has evolved differently from most gene families, with mutations accumulating most rapidly in intron regions, less rapidly in protein-conding regions, and least rapidly in 5 and 3 untranslated regions.     

The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5′-untranslated region (5′-UTR) of the mouse mammary tumor virus (MMTV). The 5′-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5′-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5′-UTR was resistant to the addition of m⁷GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5′-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.     

KRAS alleles: The LCS6 3′UTR variant and KRAS coding sequence mutations in the NCI-60 panel

The KRAS-variant is a germline single nucleotide polymorphism (SNP) within the 3′ UTR of the KRAS gene predicted to disrupt a complementary binding site (LCS6) for the let-7 microRNA (miRNA). The KRAS-variant is associated with increased risk of various cancers, including lung cancer, ovarian cancer and triple-negative breast cancer, and is associated with altered tumor biology in head and neck cancer, colon cancer and melanoma. To better understand the molecular pathways that may be regulated or affected by the presence of the KRAS-variant allele in cancer cells, we examined its prevalence in the NCI-60 panel of cell lines and sought to identify common features of the cell lines that carry the variant allele. This study provides a step forward towards understanding the molecular and pathological significance of the KRAS-variant.     

Heterogeneity in 5′-nucleotidase activity of mouse peritoneal macrophages

Summary After stimulation of the mouse peritoneal cavity with newborn calf serum (NBCS), four types of monocyte and macrophage were distinguished on the basis of peroxidase (PO) patterns. Cytochemically, these cells showed strong heterogeneity in 5-nucleotidase (5N) activity. Monocytes and monocyte-derived macrophages with PO activity in granules lacked 5N activity. Resident macrophages (with PO activity in RER and nuclear envelope) generally had significant 5N activity on the plasma membrane, the pattern showing close correlation with the biochemical findings. The group of PO-negative macrophages comprised both 5N-negative and 5N-positive cells. These findings suggest two possibilities, i.e., that monocytes (5N-)transform via PO-negative cells (5N-/+) into resident macrophages (5N+), or that the monocytes and monocyte-derived macrophages and the resident macrophages represent separate lineages. The fourth type of macrophage, the exudate-resident cell (with PO activity both in granules and in the RER and nuclear envelope), occurred only in low numbers and very late after NBCS stimulation, and is therefore considered not to be a transitional cell between monocytes and resident macrophages.     

Transposition of Tnr1 in rice genomes to 5′-PuTAPy-3′ sites,duplicating the TA sequence

Tnr1 is a repetitive sequence in rice with several features characteristic of a transposable DNA element. Its copy number was estimated to be about 3500 per haploid genome by slot-blot hybridization. We have isolated six members of Tnr1 located at different loci by PCR (polymerase chain reaction) and determined their nucleotide sequences. The Tnr1 elements were similar in size and highly homologous (about 85%) to the Tnr1 sequence identified first in the Waxy gene in Oryza glaberrima. A consensus sequence of 235 by could be derived from the nucleotide sequences of all the Tnr1 members. The consensus sequence showed that base substitutions occurred frequently in Tnr1 by transition, and that Tnr1 has terminal inverted repeat sequences of 75 bp. Almost all the chromosomal sequences that flank the Tnr1 members were 5′-PuTA-3′ and 5′-TAPy-3′, indicating that Tnr1 transposed to 5′-PuTAPy-3′ sites, duplicating the TA sequence. PCR-amplified fragments from some rice species did not contain the Tnr1 members at corresponding loci. Comparison of nucleotide sequences of the fragments with or without a Tnr1 member confirmed preferential transposition of Tnr1 to 5′-PuTAPy-3′ sites, duplicating the TA sequence. One amplified sequence suggested that imprecise excision had occurred to remove a DNA segment containing a Tnr1 member and its neighboring sequences at the Waxy locus of rice species with genome types other than AA. We also present data that may suggest that Tnr1 is a defective form of an autonomous transposable element.     

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 5′ UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3′ UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 3′ end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3′ end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA–RNA interaction between the 5′ and 3′ ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 5′–3′ end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.