Practical Aspects of microRNA Target Prediction.

microRNAs (miRNAs) are endogenous non-coding RNAs that control gene expression at the posttranscriptional level. These small regulatory molecules play a key role in the majority of biological processes and their expression is also tightly regulated. Both the deregulation of genes controlled by miRNAs and the altered miRNA expression have been linked to many disorders, including cancer, cardiovascular, metabolic and neurodegenerative diseases. Therefore, it is of particular interest to reliably predict potential miRNA targets which might be involved in these diseases. However, interactions between miRNAs and their targets are complex and very often there are numerous putative miRNA recognition sites in mRNAs. Many miRNA targets have been computationally predicted but only a limited number of these were experimentally validated. Although a variety of miRNA target prediction algorithms are available, results of their application are often inconsistent. Hence, finding a functional miRNA target is still a challenging task. In this review, currently available and frequently used computational tools for miRNA target prediction, i.e., PicTar, TargetScan, DIANA-microT, miRanda, rna22 and PITA are outlined and various practical aspects of miRNA target analysis are extensively discussed. Moreover, the performance of three algorithms (PicTar, TargetScan and DIANA-microT) is both demonstrated and evaluated by performing an in-depth analysis of miRNA interactions with mRNAs derived from genes triggering hereditary neurological disorders known as trinucleotide repeat expansion diseases (TREDs), such as Huntington's disease (HD), a number of spinocerebellar ataxias (SCAs), and myotonic dystrophy type 1 (DM1).

Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection.

SSCP and heteroduplex analysis (HA) continue to be the most popular methods of mutation detection due to their simplicity, high sensitivity and low cost. The advantages of these methods are most clearly visible when large genes, such as BRCA1 and BRCA2, are scanned for scattered unknown mutations and/or when a large number of DNA samples is screened for specific mutations. Here we describe a novel combined SSCP/duplex analysis adapted to the modern capillary electrophoresis (CE) system, which takes advantage of multicolor labeling of DNA fragments and laser-induced fluorescence detection. In developing this method, we first established the optimum conditions for homoduplex and heteroduplex analysis by CE. These were determined based on comprehensive analysis of representative Tamra-500 markers and BRCA1 fragments at different concentrations of sieving polymer and temperatures in the presence or absence of glycerol. The intrinsic features of DNA duplex structures are discussed in detail to explain differences in the migration rates between various types of duplexes. When combined SSCP/duplex analysis was carried out in single conditions, those found to be optimal for analysis of duplexes, all 31 BRCA1 and BRCA2 mutations, polymorphisms and variants tested were detected. It is worth noting that the panel of analyzed sequence variants was enriched in base substitutions, which are usually more difficult to detect. The sensitivity of mutation detection in the SSCP portion alone was 90%, and that in the duplex portion was 81% in the single conditions of electrophoresis. As is also shown here, the proposed combined SSCP/duplex analysis by CE has the potential of being applied to the analysis of pooled genomic DNA samples, and to multiplex analysis of amplicons from different gene fragments. These modifications may further reduce the costs of analysis, making the method attractive for large scale application in SNP scanning and screening.

BRCA2 germline mutations in male breast cancer patients in the Polish population.

Breast cancer is a rare disease in men. Germ-line mutations in BRCA2 and androgen receptor (AR) genes are thought to be responsible for a proportion of male breast cancer cases. The present study was performed on a series of 37 consenting patients not selected for family history of breast/ovarian cancer. The entire coding region of the BRCA2 gene and two exons of the AR gene were analyzed for germ-line mutations to evaluate the association between BRCA2 and AR genes and male breast cancer in Poland. We identified four frameshift mutations (11%) in exons 10, 11, 17 and 18, two of them were novel: 6495del3insC and 8457insA. Three missense unclassified variants (8%) of the BRCA2 gene were also identified. The frequencies of missense alterations were examined in a set of 200 chromosomes. No alteration of the AR gene was found. We did not observe much difference in clinicopathological features between carriers and non-carriers of BRCA2 mutations. Five of 37 patients (14%) had a family history of breast cancer, in one first- or second-degree relative, among the latter was one mutation carrier. The results of this study suggest that germ-line BRCA2 mutations account for rather small proportion of male breast cancer in Poland.

Prevalence of BRCA1 founder mutations in western Poland.

The frequency of three BRCA1 founder mutations was examined in a group of Polish breast/ovarian cancer patients and women at increased risk of these cancers based on family history. Among the 15 mutations found (5385-5386insC, 187-188delAG, and 4154delA), eight were detected in women with a low or moderate family history. The frequency of the IVS20+48ins12 variant was also analyzed and its distribution within risk groups argues against its involvement in cancer predisposition.

Allelic imbalance of BRCA1 transcript in the IVS20 12-bp insertion carrier.

One of the unclassified variants of the BRCA1 gene which has drawn considerable attention in recent years is the 12-bp insertion/duplication in intron 20. In this report, we show that a contribution from one chromosome cannot be detected in the BRCA1 transcript of the 12 bp insertion carrier. We also demonstrate here that the single transcript variant we observe by cDNA analysis originates from the same BRCA1 allele that harbours the 12-bp insertion. Hum Mutat 16:371, 2000.

High frequency of recurrent mutations in BRCA1 and BRCA2 genes in Polish families with breast and ovarian cancer.

Germ-line mutations in BRCA1 and BRCA2 genes result in a significantly increased risk of breast and ovarian cancer. Other genes involved in an increased predisposition to breast cancer include the TP53 gene, mutated in Li-Fraumeni syndrome. To estimate the frequency of germ-line mutations in these three genes in Upper Silesia, we have analyzed 47 breast/ovarian cancer families from that region. We found five different disease predisposing mutations in 17 (36%) families. Twelve families (25.5%) carried known BRCA1 mutations (5382insC and C61G), four families (8.5%) carried novel BRCA2 mutations (9631delC and 6886delGAAAA), and one family (2%) harbored novel mutation 1095del8 in the TP53 gene, which is the largest germline deletion in coding sequence of this gene identified thus far. The 5382insC mutation in BRCA1 was found in 11 families and the 9631delC mutation in BRCA2 occurred in three families. These two mutations taken together contribute to 82% of all mutations found in this study, and 30% of the families investigated harbor one of these mutations. The very high frequency of common mutations observed in these families can only be compared to that reported for Ashkenazi Jewish, Icelandic, and Russian high-risk families. This frequency, however, may not be representative for the entire Polish population. The observed distribution of mutations will favor routine pre-screening of predisposed families using a simple and cost-effective test.

Patterns of cleavages induced by lead ions in defined RNA secondary structure motifs.

We have characterized the susceptibility of various RNA bulges, loops and other single-stranded sequences to hydrolysis promoted by Pb2+. The reactivity of bulges depends primarily on the structural context of the flanking base-pairs and the effect of nucleotide present at the 5' side of the bulge is particularly strong. The efficiency of stacking interactions between the bulged residue and its neighbors seems to determine cleavage specificity and efficiency. Hydrolysis of two- and three-nucleotide bulges depends only slightly on their nucleotide composition. In the case of terminal loops, the efficiency of their hydrolysis usually increases with the loop size and strongly depends on its nucleotide composition. Stable tetraloops UUCG, CUUG and GCAA are resistant to hydrolysis, while in some other loops of the GNRA family a single, weak cleavage occurs, suggesting the existence of structural subclasses within the family. A very efficient, specific hydrolysis of a phosphodiester bond in the single-stranded region adjacent to the stem in oligomer 12 resembles highly specific cleavages of some tRNA molecules. The reaction occurs in the presence of Pb2+, but not in the presence of several other metal ions. The Pb(2+)-cleavable RNA domain may be considered another example of leadzyme. The results of Pb(2+)-induced hydrolysis in model RNA oligomers should be useful in interpretation of cleavage patterns of much larger, naturally occurring RNA molecules.

CUG repeats present in myotonin kinase RNA form metastable "slippery" hairpins.

We show that CUG repeats form "slippery" hairpins in their natural sequence context of the myotonin kinase gene transcript. This novel type of RNA structure is characterized by strong S1 and T1 nuclease and lead cleavages in the terminal loop and by mild lead cleavages in the hairpin stem. The latter effect indicates a relaxed metastable structure of the stem. (CUG)5 repeats do not form any detectable secondary structure, whereas hairpins of increasing stability are formed by (CUG)11, (CUG)21, and (CUG)49. The potential role of the RNA hairpin structure in the pathogenesis of myotonic dystrophy is discussed.

Structural analysis of two plant 5S rRNA species and fragments thereof by lead-induced hydrolysis.

The structure of plant 5S rRNA species from lupin and wheat germ as well as the structure of two RNA fragments that represent domains beta and gamma of lupin 5S rRNA have been probed by Pb(II)-induced hydrolysis. The lead digestion patterns of 5S rRNA species show that the secondary and tertiary structures of the molecules are very similar. The data suggests that two potential base pairs at the bottom of helix E are destabilized and this causes an enlargement of the hairpin loop e. On the other hand, nucleotides from loop c seem to be involved in the formation of some kind of higher order structure. A comparison of the distribution of cleavages induced in RNA fragments to those in the corresponding regions of the entire 5S rRNA shows that under conditions applied in our studies the structural domains beta and gamma are not involved in formation of any tertiary interaction within 5S rRNA structure.

Effect of modified nucleotides on structure of yeast tRNA(Phe). Comparative studies by metal ion-induced hydrolysis and nuclease mapping.

Structural differences between native yeast tRNA(Phe), its in vitro transcript and the U8G mutant have been investigated using metal ion-induced hydrolysis and nuclease digestion. Differences in the solution structure of the molecules involve four regions: the D- and T-loops, the variable region and the anticodon loop. Efficiency of the Pb(II); Eu(II)-, Mn(II)- and Mg(II)-induced hydrolysis at the main cleavage sites in the D-loop is significantly reduced for unmodified tRNAs. Moreover, only the in vitro transcripts are susceptible for cleavage in the T-loop and entire anticodon loop. Other changes in the transcript molecule involve 50-fold enhancement of S1 nuclease digestion at p36, weak cleavages in the D-loop and lack of some digestion sites in the T-loop. The nuclease V1 digestion patterns are very similar for studied molecules. Changes in the pattern of hydrolysis of the D-loop caused by mutation of the conservative base U8 to G are detected by metal-induced hydrolysis only. Our results indicate clearly that metal ions and enzymatic probes monitor different features of RNA structure and their combined use is highly advantageous in studying subtle structural changes in tRNA.

Specific RNA cleavages induced by manganese ions.

The specificity and efficiency of manganese ion-induced RNA hydrolysis was studied with several tRNA molecules. In case of yeast tRNA(Phe), the main cleavage occurs at p16 and minor cuts at p17-18, p20-21, p34 and p36-37. The major Mn(II)-induced cut in yeast elongator tRNA(Met) is also located in the D-loop at p16 and it is stronger than that observed in tRNA(Phe). In initiator tRNA(Met) from yeast two strong Mn(II) cleavages of equal intensity occur at p16 and p17. This is in contrast with single, much weaker cuts induced in the D-loop of that tRNA by Mg(II), Eu(III) and Pb(II) ions. Interestingly, in case of yeast tRNA(Glu) the main cleavage caused by Mn(II), Mg(II) and Pb(II) ions occurs in the anticodon loop. The involvement of hypermodified base mnm5s2U in this cleavage was ruled out based on results obtained with in vitro transcript of yeast tRNA(Glu) anticodon arm. Mutation of a single base A37G in the anticodon loop of the transcript drastically reduced the specificity of Mn(II)-induced hydrolysis.

Faster and cheaper PCR on a standard thermocycler.

The PCR conditions have been optimized to make the process faster and more economical. When short DNA fragments are to be amplified, the time of denaturation, annealing and extension steps can be as short as 1 s each, and the yield of PCR product is still high, sufficient for many types of analysis. The PCR can be done even in a reaction volume as low as 1 microliter. The recommended volume, 2.5 microliters or 5 microliters, allows significant savings in the laboratory budget especially for laboratories which use PCR frequently and on a large scale.

Isolation of genes specifically expressed in flat revertant cells derived from activated ras-transformed NIH 3T3 cells by treatment with azatyrosine.

We previously reported that mouse NIH 3T3 cells transformed by transfection of activated human c-Ha-ras become apparently normal upon treatment with the antibiotic azatyrosine. The revertant cells maintain their normal phenotype during prolonged culture in the absence of azatyrosine, although activated p21ras is still expressed. The normal phenotype induced by azatyrosine could be due to activation of expression of some cellular gene(s) in the cells that results in suppression of ras function. To identify the genes with increased expression in the revertant cells, we adopted differential screening of recombinants from a phage cDNA library made from mRNA of the revertant cells, hybridized with 32P-labeled cDNAs made from mRNAs of the ras-transformed NIH 3T3 cells and the revertant cells. Two clones thus isolated were found to be almost identical to the ras recision gene (rrg), which was identified as a tumor-suppressor gene by Contente et al. [Contente, S., Kenyon, K., Rimoldi, D. & Friedman, R. M. (1990) Science 249, 796-798]. Other genes identified were the collagen type III and rhoB genes. Approximately half the clones were found to contain a sequence corresponding to that of the murine retrovirus-like intracisternal A particle. We speculate that azatyrosine activates several cellular genes in the ras-transformed cells and that some of these genes, including rrg, act cooperatively to counteract ras function, resulting in reversion of the ras-transformed cells to the normal phenotype.

Analysis of magnesium, europium and lead binding sites in methionine initiator and elongator tRNAs by specific metal-ion-induced cleavages.

The specificity of cleavages in yeast and lupin initiator and elongator methionine tRNAs induced by magnesium, europium and lead has been analysed and compared with known patterns of yeast tRNA(Phe) hydrolysis. The strong D-loop cleavages occur in methionine elongator tRNAs at similar positions and with comparable efficiency to those found in tRNA(Phe), while the sites of weak anticodon loop cuts, identical in methionine elongator tRNAs, differ from those found in tRNA(Phe). Methionine initiator tRNAs differ from their elongator counterparts: (a) they are cleaved in the D-loop with much lower efficiency; (b) they are cleaved in the variable loop which is completely resistant to hydrolysis in elongator tRNAs; (c) cleavages in the anticodon loop are stronger in initiator tRNAs and they are located mostly at the 5' side of the loop whereas in elongator tRNAs they occur mostly at the opposite, 3' side of the loop. The distinct pattern of the anticodon loop cleavages is considered to be related to different conformations of the anticodon loop in the two types of methionine tRNAs.

Specificity and mechanism of the cleavages induced in yeast tRNAPhe by magnesium ions.

The specificity of magnesium ion-induced hydrolysis of yeast tRNAPhe in solution was studied as a function of the excess of Mg(II) ions and pH. The major cuts at phosphates 16 and 20 as well as minor cleavages at phosphates 17, 18, 21, 34 and 36 occur at all pH values in the range of 8.0-9.5, and at a molar excess of magnesium ions over the tRNA ranging from 125 to 5000. In yeast tRNA(Phe)-Y the efficiency of the anticodon and D-loop cleavages is considerably decreased while the differently modified Y-base of yellow lupin tRNA(Phe) lowers the specificity of the weak anticodon loop cleavages. The mechanism of the Mg(II)-induced cleavages is discussed on the basis of yeast tRNA(Phe) crystal structure data, and the two major D-loop cleavages are thought to be effected from two distinct magnesium binding sites. The possibility of probing the environments of magnesium binding sites in tRNAs by the induced cleavages is demonstrated, and the relevance of magnesium-induced tRNA cleavages to RNA catalysis is discussed.

Identification of the magnesium, europium and lead binding sites in E. coli and lupine tRNAPhe by specific metal ion-induced cleavages.

The Pb, Eu and Mg-induced cleavages in E. coli and lupine tRNAPhe have been characterized and compared with those found in yeast tRNAPhe. The pattern of lupine tRNAPhe hydrolysis closely resembles that of yeast tRNAPhe, while several major differences occur in the specificity and efficiency of the E. coli tRNAPhe hydrolysis. The latter tRNA is cleaved with much lower yield in the D-loop, and interestingly, cleavage is also detected in the variable region, that is highly resistant to hydrolysis in eukaryotic tRNAs. The possible location of tight Pb, Eu and Mg binding sites in E. coli tRNAPhe is discussed on the basis of the specific hydrolysis data.

An efficiently mutagenizable recombinant plasmid for in vitro transcription of the Escherichia coli 16 S RNA gene.

The portion of the rrnB operon coding for 16 S RNA was modified to permit efficient in vitro transcription by T7 RNA polymerase of full-length, correctly terminated, biologically active 16 S RNA (W. Krzyzosiak et al., 1987, Biochemistry 26, 2353-2364). The 5'-end of the gene was fused to the class III T7 promoter and the 3'-end was modified so that cleavage with MstII would generate correctly terminated RNA upon runoff transcription. The modified gene was placed in pUC19 by a four-way ligation reaction involving linearized pUC19, a 1490-bp fragment of 16 S rDNA, and two synthetic oligodeoxynucleotides. Because of the cohesive end design, phosphorylation of the synthetic oligomers was not necessary. Single and tandem cassette insertions were used to generate single base changes in the C-1400 region of 16 S RNA. Three examples are described. This method is generally applicable to the 16 S RNA molecule as suitable singlecleavage restriction sites allow all regions to be mutated by this approach.

Characterization of the lead(II)-induced cleavages in tRNAs in solution and effect of the Y-base removal in yeast tRNAPhe.

The specificity of lead(II)-induced hydrolysis of yeast tRNA(Phe) was studied as a function of concentration of Pb2+ ions. The major cut was localized in the D-loop and minor cleavages were detected in the anticodon and T-loops at high metal ion concentration. The effects of pH, temperature, and urea were also analyzed, revealing a basically unchanged specificity of hydrolysis. In the isolated 5'-half-molecule of yeast tRNAPhe not cut was found in the D-loop, indicating its stringent dependence on T-D-loop interaction. Comparison of hydrolysis patterns and efficiencies observed in yeast tRNA(Phe) with those found in other tRNAs suggests that the presence of a U59-C60 sequence in the T-loop is responsible for the highly efficient and specific hydrolysis in the spatially close region of the D-loop. The efficiencies of D-loop cleavage in intact yeast tRNA(Phe) and in tRNA(Phe) deprived of the Y base next to the anticodon were also compared at various Pb2+ ion concentrations. Kinetics of the D-loop hydrolysis analyzed at 0, 25, and 37 degrees C showed a 6 times higher susceptibility of tRNA(Phe) minus Y base (tRNA(Phe)-Y) to lead(II)-induced hydrolysis than in tRNA(Phe). The observed effect is discussed in terms of a long-distance conformational transition in the region of the interacting D- and T-loops triggered by the Y-base excision.