Type I collagen is the most abundant protein in human body, produced by folding of two α1(I) and one α2(I) polypeptides into the triple helix. A conserved stem-loop structure is found in the 5' UTR of collagen mRNAs, encompassing the translation start codon. We cloned La ribonucleoprotein domain family, member 6 (LARP6) as the protein which binds the collagen 5' stem-loop in the sequence specific manner. LARP6 has a distinctive bipartite RNA binding domain, not found in other members of the La superfamily. LARP6 interacts with the two single stranded regions of 5' stemloop. The Kd for binding of LARP6 to the 5' stem-loop is 1.4 nM. LARP6 binds the 5' stem-loop in both, the nucleus and cytoplasm. In the cytoplasm, LARP6 does not associate with polysomes, however, overexpression of LARP6 blocks ribosomal loading on collagen mRNAs. Knocking down LARP6 by siRNA also decreased polysomal loading of collagen mRNAs, suggesting that it regulates translation. Collagen protein is synthesized at discrete regions of the endoplasmic reticulum (ER). We could reproduce this focal pattern of synthesis using collagen/GFP reporter protein, but only when the reporter was encoded by the mRNA with the 5' stem-loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5' stem-loop, or in absence of LARP6, it accumulated diffusely throughout the ER. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulate that LARP6 dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer.

We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase ␦) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1-to 8-bp repeat units). Both the rad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the "proofreading" exonuclease domain of DNA polymerase ␦) mutations were synthetically lethal.All eukaryotic genomes thus far examined contain many simple repetitive DNA sequences, tracts of DNA with one or a small number of bases repeated multiple times (48). These repetitive regions can be classified as microsatellites (small repeat units in tandem arrays 10 to 60 bp in length) and minisatellites (larger repeat units in tandem arrays several hundred base pairs to several kilobase pairs in length). In this paper, arrays with repeat units 14 bp or less will be considered microsatellites and arrays with longer repeat units will be considered minisatellites.Previous studies show that simple repetitive sequences are unstable relative to "normal" DNA sequences, frequently undergoing additions or deletions of repeat units, in Escherichia coli (24), Saccharomyces cerevisiae (12), and mammals (59). This mutability has two important consequences. First, it results in polymorphic loci that are useful in genetic mapping and forensic studies (15,59). Second, although these repetitive tracts are usually located outside of coding sequences, alterations in the lengths of microsatellites or minisatellites located within coding sequences can produce frameshift mutations or novel protein variants (20,22,26).From studies of the effects of various mutations on microsatellite stability in yeast and E. coli (40) and the analysis of mutational changes caused by DNA polymerase in vitro (21), it is likely that most alterations reflect DNA polymerase slippage events (47). These events involve the transient dissociation of the primer and template strands during the replication of a microsatellite (Fig. 1). If the strands reassociate to yield an unpaired repeat on the primer strand, the net result is an addition of repeats (following a second round of DNA replication). Unpaired repeats on the template strand would result in a deletion by the same mechanism.A number of mutations have been shown to elevate microsatellite instability. In E. coli (24, 46), yeast (44, 45), and mammalian cells (27), mutations in genes affecting DNA mismatch repair dramatically elevate the ins...

In Saccharomyces cerevisiae, POL3 encodes the catalytic subunit of DNA polymerase ␦. While yeast POL3 mutant strains that lack the proofreading exonuclease activity of the polymerase have a strong mutator phenotype, little is known regarding the role of other Pol3p domains in mutation avoidance. We identified a number of pol3 mutations in regions outside of the exonuclease domain that have a mutator phenotype, substantially elevating the frequency of deletions. These deletions appear to reflect an increased frequency of DNA polymerase slippage. In addition, we demonstrate that reduction in the level of wild-type DNA polymerase results in a similar mutator phenotype. Lowered levels of DNA polymerase also result in increased sensitivity to the DNA-damaging agent methyl methane sulfonate. We conclude that both the quantity and the quality of DNA polymerase ␦ is important in ensuring genome stability.The low mutation rate observed in wild-type cells reflects both the accuracy of DNA polymerases and the existence of DNA repair systems that remove misincorporated bases. Mutations affecting components of either of these systems can result in a mutator phenotype, a global elevation in mutation frequencies throughout the genome (27). In the yeast Saccharomyces cerevisiae, a mutator phenotype has been associated with certain mutations of POL3 and POL2, encoding the replicative DNA polymerases ␦ and ε, respectively (12, 22, 37, 55); these alleles can reside in either a DNA-proofreading exonuclease domain of POL3 (Exo I domain [ Fig. 1]) or near domains required for nucleotide binding (domains II and VI). Certain mutant substitutions of POL30, encoding the DNA polymerase processivity factor PCNA, also have a strong mutator phenotype (8,20,23,56). In addition, null mutations of RAD27 (encoding an Okazaki fragment-processing enzyme) or certain alleles of RPA1 (encoding the large subunit of a singlestranded DNA binding protein) substantially elevate mutation rates (7,19,22,54). A number of mutants in Schizosaccharomyces pombe, including those affecting DNA polymerases ␣ and ␦ and DNA ligase, also exhibit increased rates of mutation (33).In addition to mutations affecting DNA replication genes, mutations of the DNA mismatch repair genes have a mutator phenotype. Most mismatch repair in yeast involves two complexes, although other complexes have minor roles (24). Basebase mismatches are corrected by a heterotetramer involving Msh2p, Msh6p, Pms1p, and Mlh1p. Small DNA loops, resulting from DNA polymerase slippage events on simple repetitive DNA sequences (microsatellites) ( Fig. 2A), are repaired by a complex that includes Msh2p, Msh3p, Pms1p, and Mlh1p. Failure to repair base-base mismatches results in an elevated frequency of single-base-pair substitutions, whereas failure to repair DNA loops results in an elevated frequency of deletions or insertions (47). Genetic and biochemical data indicate that the Msh2p-Msh3p Pms1p-Mlh1p complex can correct DNA loops up to 14 bases in size but is incapable of correcting loops that are 16 ...

Evolutionary studies have suggested that mutation rates vary significantly at different positions in the eukaryotic genome. The mechanism that is responsible for this context-dependence of mutation rates is not understood. We demonstrate experimentally that frameshift mutation rates in yeast microsatellites depend on the genomic context and that this variation primarily reflects the context-dependence of the efficiency of DNA mismatch repair. We measured the stability of a 16.5-repeat polyGT tract by using a reporter gene (URA3-GT) in which the microsatellite was inserted in-frame into the yeast URA3 gene. We constructed 10 isogenic yeast strains with the reporter gene at different locations in the genome. Rates of frameshift mutations that abolished the correct reading frame of this gene were determined by fluctuation analysis. A 16-fold difference was found among these strains. We made mismatch-repair-deficient (msh2) derivatives of six of the strains. Mutation rates were elevated for all of these strains, but the differences in rates among the strains were substantially reduced. The simplest interpretation of this result is that the efficiency of DNA mismatch repair varies in different regions of the genome, perhaps reflecting some aspect of chromosome structure.genetic instability ͉ microsatellite ͉ mutation rate ͉ Saccharomyces cerevisiae C omparisons of amino acid or base sequences of orthologous genes indicate that different genes evolve at different rates (1). Differences in the rates of accumulation of amino acid changes or nonsynonymous base substitutions are influenced by selective constraints (1). For highly expressed genes, the rate of synonymous base substitutions is affected by GC content and codon bias (2, 3). In Saccharomyces cerevisiae and mammalian cells, the rates of synonymous substitutions also vary by a factor of Ϸ10, depending on the position of the gene in the genome (4-6). The interpretation of these observations is unclear. It is possible that the misincorporation rates of the replicative DNA polymerases are different at different positions in the genome. Alternatively, the fidelity of DNA polymerases may be invariant, but the detection and repair of misincorporation events may be context-specific.Microsatellites are regions of DNA in which a single base or a small number of bases is repeated in tandem. The polyGT sequence is a particularly common microsatellite in many eukaryotes (7). We have developed (8, 9) methods of measuring the rate of microsatellite alterations. In this study, we used this assay to measure the mutation rates of the same polyGT microsatellite placed in 10 different chromosomal contexts in the yeast genome. We show that the microsatellite mutation rates vary by more than an order of magnitude among different genomic positions in yeast strains that have wild-type DNA mismatch repair. We have demonstrated (8) that the mutation rates of microsatellites are greatly elevated in yeast strains with deficient mismatch repair. In this study, we find that microsatellite in...

Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes. Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts. Activated HSCs are collagen-producing cells in the fibrotic liver. Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50-to 70-fold. Likewise, expression of TRAM2 dramatically increases in activated HSCs. TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon. However, TRAM2 has a C terminus with only a 15% identity. The C-terminal part of TRAM2 interacts with the Ca 2؉ pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells. TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact. Deletion of the Cterminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs. The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect. Depletion of ER Ca 2؉ with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation. We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon. This coupling may increase the local Ca 2؉ concentration at the site of collagen synthesis, and a high Ca 2؉ concentration may be necessary for the function of molecular chaperones involved in collagen folding.