Facile FMR1 mRNA structure regulation by interruptions in CGG repeats

RNA metabolism is a major contributor to the pathogenesis of clinical disorders associated with premutation size alleles of the fragile X mental retardation (FMR1) gene. Herein, we determined the structural properties of numerous FMR1 transcripts harboring different numbers of both CGG repeats and AGG interruptions. The stability of hairpins formed by uninterrupted repeat-containing transcripts increased with the lengthening of the repeat tract. Even a single AGG interruption in the repeated sequence dramatically changed the folding of the 5′UTR fragments, typically resulting in branched hairpin structures. Transcripts containing different lengths of CGG repeats, but sharing a common AGG pattern, adopted similar types of secondary structures. We postulate that interruption-dependent structure variants of the FMR1 mRNA contribute to the phenotype diversity, observed in premutation carriers

Mutant CAG repeats of Huntingtin transcript fold into hairpins, form nuclear foci and are targets for RNA interference

The CAG repeat expansions that occur in translated regions of specific genes can cause human genetic disorders known as polyglutamine (poly-Q)-triggered diseases. Huntington’s disease and spinobulbar muscular atrophy (SBMA) are examples of these diseases in which underlying mutations are localized near other trinucleotide repeats in the huntingtin (HTT) and androgen receptor (AR) genes, respectively. Mutant proteins that contain expanded polyglutamine tracts are well-known triggers of pathogenesis in poly-Q diseases, but a toxic role for mutant transcripts has also been proposed. To gain insight into the structural features of complex triplet repeats of HTT and AR transcripts, we determined their structures in vitro and showed the contribution of neighboring repeats to CAG repeat hairpin formation. We also demonstrated that the expanded transcript is retained in the nucleus of human HD fibroblasts and is colocalized with the MBNL1 protein. This suggests that the CAG repeats in the HTT mRNA adopt ds-like RNA conformations in vivo. The intracellular structure of the CAG repeat region of mutant HTT transcripts was not sufficiently stable to be protected from cleavage by an siRNA targeting the repeats and the silencing efficiency was higher for the mutant transcript than for its normal counterpart

Trinucleotide repeats in human genome and exome

Trinucleotide repeats (TNRs) are of interest in genetics because they are used as markers for tracing genotype–phenotype relations and because they are directly involved in numerous human genetic diseases. In this study, we searched the human genome reference sequence and annotated exons (exome) for the presence of uninterrupted triplet repeat tracts composed of six or more repeated units. A list of 32 448 TNRs and 878 TNR-containing genes was generated and is provided herein. We found that some triplet repeats, specifically CNG, are overrepresented, while CTT, ATC, AAC and AAT are underrepresented in exons. This observation suggests that the occurrence of TNRs in exons is not random, but undergoes positive or negative selective pressure. Additionally, TNR types strongly determine their localization in mRNA sections (ORF, UTRs). Most genes containing exon-overrepresented TNRs are associated with gene ontology-defined functions. Surprisingly, many groups of genes that contain TNR types coding for different homo-amino acid tracts associate with the same transcription-related GO categories. We propose that TNRs have potential to be functional genetic elements and that their variation may be involved in the regulation of many common phenotypes; as such, TNR polymorphisms should be considered a priority in association studies

RNA structure of trinucleotide repeats associated with human neurological diseases

The tandem repeats of trinucleotide sequences are present in many human genes and their expansion in specific genes causes a number of hereditary neurological disorders. The normal function of triplet repeats in transcripts is barely known and the role of expanded RNA repeats in the pathogenesis of Triplet Repeat Expansion Diseases needs to be more fully elucidated. Here we have described the structures formed by transcripts composed of AAG, CAG, CCG, CGG and CUG repeats, which were determined by chemical and enzymatic structure probing. With the exception of the repeated AAG motif, all studied repeats form hairpin structures and these hairpins show several alternative alignments. We have determined the molecular architectures of these co-existing hairpin structures by using transcripts with GC-clamps which imposed single alignments of hairpins. We have provided experimental evidence that CCUG repeats implicated in myotonic dystrophy type 2 also form hairpin structures with properties similar to that composed of the CUG repeats

SERPINA3: Stimulator or Inhibitor of Pathological Changes

SERPINA3, also called α-1-antichymotrypsin (AACT, ACT), is one of the inhibitors of serine proteases, one of which is cathepsin G. As an acute-phase protein secreted into the plasma by liver cells, it plays an important role in the anti-inflammatory response and antiviral response. Elevated levels of SERPINA3 have been observed in heart failure and neurological diseases such as Alzheimer’s disease or Creutzfeldt–Jakob disease. Many studies have shown increased expression levels of the SERPINA3 gene in various types of cancer, such as glioblastoma, colorectal cancer, endometrial cancer, breast cancer, or melanoma. In this case, the SERPINA3 protein is associated with an antiapoptotic function implemented by adjusting the PI3K/AKT or MAPK/ERK 1/2 signal pathways. However, the functions of the SERPINA3 protein are still only partially understood, mainly in the context of cancerogenesis, so it seems necessary to summarize the available information and describe its mechanism of action. In particular, we sought to amass the existing body of research focusing on the description of the underlying mechanisms of various diseases not related to cancer. Our goal was to present an overview of the correct function of SERPINA3 as part of the defense system, which unfortunately easily becomes the “Fifth Column” and begins to support processes of destruction

Structure analysis of 5′UTR fragments of the mRNA containing CGG repeats with single AGG interruption

<p><b>Copyright information:</b></p><p>Taken from "Facile FMR1 mRNA structure regulation by interruptions in CGG repeats"</p><p>Nucleic Acids Research 2005;33(2):451-463.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548340.</p><p>© 2005, the authors © </p> () Native polyacrylamide gel electrophoresis of the 5′ labeled transcripts. Arrowheads indicate coexisting stable slow migrating (S) and fast migrating (F) conformers. The ds69 and ds107 represent dsRNA migration standards (see Materials and Methods). (–) Cleavage patterns obtained for 5′ end labeled conformers S and F of the fx4 transcript treated with: Pb(II), lanes 1 and 2 correspond to 0.25 and 1.0 mM lead acetate, respectively; S, lanes 1 and 2 correspond to 0.675 and 2.7 U/μl of S nuclease, respectively; T, lanes 1 and 2 correspond to 0.1 and 0.5 U/μl of T ribonuclease, respectively; U, lanes 1 and 2 correspond to 0.5 and 1 U/μl of U ribonuclease; lane C, incubation control; L, formamide ladder; Cl, cytosine-specific ladder; U, adenine-specific ladder. The first and the last cytosine of the CGG tract are indicated, as well as the adenine residue of the AGG interruption. The regions of the RNA molecules predicted to form single-stranded loops are shown. Electrophoresis was conducted in a 10% denaturing polyacrylamide gel. () Cleavage patterns obtained for 5′ end labeled conformers S and F of the fx5 transcript. The reaction conditions were the same as indicated in the panel B and C. () Results of the enzymatic probing of the 5′ end labeled fx6 transcript. The reaction conditions were the same as indicated in the panel B and C

Structure analysis of 5′UTR fragments of the mRNA containing CGG repeats with two AGG interruptions

<p><b>Copyright information:</b></p><p>Taken from "Facile FMR1 mRNA structure regulation by interruptions in CGG repeats"</p><p>Nucleic Acids Research 2005;33(2):451-463.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548340.</p><p>© 2005, the authors © </p> () Cleavage patterns obtained for 5′ end labeled conformers S and F of the fx7 transcript harboring 28 CGG repeats with two AGG interruptions treated with Pb(II), S nuclease and T ribonuclease. The reaction conditions were the same as indicated in . The first and the last cytosines of the CGG tract, as well as both the adenine residues of the AGG interruptions are indicated. The regions corresponding to the loops , and are marked. Electrophoresis was conducted in a 10% denaturing polyacrylamide gel. () Cleavage patterns obtained for both the S and F conformers of the fx7 with: P, lanes 1–3 correspond to 5, 7.5 and 10 μg/ml of P nuclease, respectively; U, lanes 1 and 2 correspond to 0.5 and 1.0 U/μl of U ribonuclease, respectively. () Cleavage patterns obtained for conformer S of the fx7 transcript digested with V ribonuclease; lanes 1–3 correspond to 4.6, 9.1 and 18.2 μ/ml of V ribonuclease. () Results of the structure probing of the fx8 transcript (conformer S) with Pb(II), S nuclease and T ribonuclease. The reactions were carried out as described in the legend to the . () Proposed secondary structure models of the 5′UTR fragments containing 28 and 30 CGG repeats with two AGG interruptions. Models of the conformers S and F are shown separately for the transcript fx7. The only difference between the structure of the fx7 and fx8 transcripts is the length of the helical region 2 (h2). Thus, for clarity, only the helix h2 terminated with the loop is shown for the fx8 transcript conformer S. It is noted that the regions flanking the CGG tract in both the models, regardless of the number of repeats, adopted identical secondary structures. Specificity and intensity of the cleavages induced by S, T and U ribonuclease are indicated. Size of the symbols corresponds to the relative cleavage intensity. Shaded area represents V ribonuclease-sensitive regions. Terminal loops of the helical regions h2 (h2′), h3 (h3′) and h4 are designated as , and , respectively. Numbers indicating the trinucleotide repeats are italicized. Below, model for the temperature and low pH induced tertiary transition from the conformer S to the conformer F of the fx7 and fx8 transcripts (see Supplementary Figure 2)

Structure analysis of 5′UTR fragments of the mRNA containing uninterrupted CGG repeats

<p><b>Copyright information:</b></p><p>Taken from "Facile FMR1 mRNA structure regulation by interruptions in CGG repeats"</p><p>Nucleic Acids Research 2005;33(2):451-463.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548340.</p><p>© 2005, the authors © </p> () Cleavage patterns obtained for 5′ end labeled fx2 transcript harboring 23 CGG repeats treated with: Pb(II), lanes 1–3 correspond to 0.25, 0.5 and 1.0 mM lead acetate, respectively; S, lanes 1–3 correspond to 0.675, 1.35 and 2.7 U/μl of S nuclease, respectively; T, lanes 1–3 correspond to 0.1, 0.3 and 0.5 U/μl of T ribonuclease, respectively; lane C, incubation control; L, formamide ladder; Cl, cytosine-specific ladder. The first and the last cytosine of the CGG tract are indicated. Electrophoresis was conducted in a 10% denaturing polyacrylamide gel. () Cleavage patterns obtained for 5′ end labeled fx3 transcript harboring 28 CGG repeats. The reaction conditions were the same as indicated in the panel A. () Cleavage patterns obtained for the terminal loops of the hairpin structures for fx1 transcript (odd number of CGG repeats) and fx3 transcript (even number of CGG repeats). Specificity and intensity of the cleavages induced by S and T nucleases are shown below. Size of the symbols corresponds to the relative cleavage intensity. () Proposed secondary structure model of the 5′UTR fragments containing 23 and 28 uninterrupted CGG repeats. It is noted that the regions flanking the CGG tract in both models, regardless of the number of repeats, adopted an identical secondary structure. The phosphodiester bonds susceptible to the lead-induced cleavages in the fx2 stem has only been shown for the 10th repeat of the hairpin for clarity; however, the same cleavage pattern was seen for all the remaining repeats of the CGG stem. The pattern of the lead ion cleavages observed in the terminal loop of fx3 is indicated. Phosphodiester bonds of the fx2 hairpin loop were very mildly cleaved by lead ions (repeats 13–15, not shown on the model). The sites of weakest cleavage induced by enzymatic probes in the CGG stem are not indicated. Numbers of the consecutive CGG repeats are italicized

SURVEY AND SUMMARY: Structures of trinucleotide repeats in human transcripts and their functional implications

Among the goals of RNA structural and functional genomics is determining structures and establishing the functions of a rich repertoire of simple sequence repeats in transcripts. These repeats are present in transcripts from their ‘birth’ in the nucleus to their ‘death’ in cytoplasm and have the potential of being involved in many steps of RNA regulation. The knowledge of their structural features and functional roles will also shed more light on the postulated mechanisms of RNA pathogenesis in a growing list of neurological diseases caused by simple sequence repeat expansions. Here, we discuss several different lines of research to support the hypothesis that the mechanism of RNA pathogenesis may be a more common phenomenon triggered or modulated also by abundant long normal repeats. We propose structures of the repeat regions in transcripts of genes involved in Triplet Repeat Expansion Diseases. We have classified the polymorphic repeat alleles of these genes according to their ability to form hairpin structures in transcripts, and describe the distribution of different structural forms of the repeats in the human population. We have also reported the results of a systematic survey of the human transcriptome to identify mRNAs containing triplet repeats and to classify them according to structural and functional criteria. Based on this knowledge, we discuss the putative wider role of triplet repeat RNA hairpins in human diseases. A hypothetical model is proposed in which long normal RNA hairpins formed by the repeats may also be involved in pathogenesis