Using RNA secondary structures to guide sequence motif finding towards single-stranded regions

RNA binding proteins recognize RNA targets in a sequence specific manner. Apart from the sequence, the secondary structure context of the binding site also affects the binding affinity. Binding sites are often located in single-stranded RNA regions and it was shown that the sequestration of a binding motif in a double-strand abolishes protein binding. Thus, it is desirable to include knowledge about RNA secondary structures when searching for the binding motif of a protein. We present the approach MEMERIS for searching sequence motifs in a set of RNA sequences and simultaneously integrating information about secondary structures. To abstract from specific structural elements, we precompute position-specific values measuring the single-strandedness of all substrings of an RNA sequence. These values are used as prior knowledge about the motif starts to guide the motif search. Extensive tests with artificial and biological data demonstrate that MEMERIS is able to identify motifs in single-stranded regions even if a stronger motif located in double-strand parts exists. The discovered motif occurrences in biological datasets mostly coincide with known protein-binding sites. This algorithm can be used for finding the binding motif of single-stranded RNA-binding proteins in SELEX or other biological sequence data

Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression

Selenium, an essential trace element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3′ untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level

RNA-binding strategies common to cold-shock domain and RNA recognition motif containing proteins.

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Interaction of nucleolin with an evolutionary conserved sequence is required for pre-rRNA primary processing in vitro.

International audienceThe first processing event of the precursor ribosomal RNA (pre-rRNA) takes place within the 5' external transcribed spacer. This primary processing requires conserved cia-acting RNA sequence downstream from the cleavage site and several nucleic acids (small nucleolar RNAs) and proteins trans-acting factors including nucleolin, a major nucleolar protein, The specific interaction of nucleolin with the pre-rRNA is required for processing in vitro. Xenopus laevis and hamster nucleolin interact with the same pre-rRNA site and stimulate the processing activity of a mouse cell extract. A highly conserved 11-nucleotide sequence located 5-6 nucleotides after the processing site is required for the interaction of nucleolin and processing. In vitro selection experiments with nucleolin have identified an RNA sequence that contains the UCGA motif present in the 11-nucleotide conserved sequence, The interaction of nucleolin with pre-rRNA is required for the formation of an active processing complex. Our findings demonstrate that nucleolin is a key factor for the assembly and maturation of pre-ribosomal ribonucleoparticles

Interaction of Nucleolin with an Evolutionarily Conserved Pre-ribosomal RNA Sequence Is Required for the Assembly of the Primary Processing Complex*

The first processing event of the precursor ribosomal RNA (pre-rRNA) takes place within the 5' external transcribed spacer. This primary processing requires conserved cia-acting RNA sequence downstream from the cleavage site and several nucleic acids (small nucleolar RNAs) and proteins trans-acting factors including nucleolin, a major nucleolar protein, The specific interaction of nucleolin with the pre-rRNA is required for processing in vitro. Xenopus laevis and hamster nucleolin interact with the same pre-rRNA site and stimulate the processing activity of a mouse cell extract. A highly conserved 11-nucleotide sequence located 5-6 nucleotides after the processing site is required for the interaction of nucleolin and processing. In vitro selection experiments with nucleolin have identified an RNA sequence that contains the UCGA motif present in the 11-nucleotide conserved sequence, The interaction of nucleolin with pre-rRNA is required for the formation of an active processing complex. Our findings demonstrate that nucleolin is a key factor for the assembly and maturation of pre-ribosomal ribonucleoparticles

Ovine ooplasm directs initial nucleolar assembly in embryos cloned from ovine, bovine, and porcine cells

Here we present ultrastructural and immunocytochemical evidence that ovine ooplasm is directing the initial assembly of the nucleolus independent of the species of the nuclear donor. Intergeneric porcine-ovine somatic cell nuclear transfer (SCNT) and intrageneric ovine-ovine SCNT embryos were constructed and the nucleolus ultrastructure and nucleolus associated rRNA synthesis examined in 1-, 2-, 4-, early 8-, late 8-, and 16-cell embryos using transmission electron microscopy (TEM) and light microscopical autoradiography. In addition, immunocytochemical localization by confocal microscopy of nucleolin, a key protein involved in processing rRNA transcripts, was performed on early 8-, late 8-, and 16-cell embryos for both groups of SCNT embryos. Intergeneric porcine-ovine SCNT embryos exhibited nucleolar precursor bodies (NPBs) of an ovine (ruminant) ultrastructure, but no active rRNA producing fibrillo-granular nucleoli at any of the stages. Unusually, cytoplasmic organelles were located inside the nucleus of two porcine-ovine SCNT embryos. The ovine-ovine SCNT embryos, on the other hand, revealed fibrillo-granular nucleoli in 16-cell embryos. In parallel, autoradiographic labeling over the nucleoplasm, and in particular, the nulcleoli was detected. Bovine-ovine SCNT embryos at the eight-cell stage were examined for nucleolar morphology and exhibited ruminant-type NPBs as well as structures that appeared as fibrillar material surrounded by a rim of electron dense granules, perhaps formerly of nucleolar origin. Nucleolin was localized throughout the nucleoplasm and with particular intensity around the presumptive nucleolar compartments for all developmental stages examined in porcine-ovine and ovine-ovine SCNT embryos. In conclusion, this study suggests that factors within the ovine ooplasm are playing a role in the initial assembly of the embryonic nucleolus in intrageneric SCNT embryos