RNA-specific nucleotidyl transferases (rNTrs) are a diverse family of template-independent polymerases that add ribonucleotides to the 3'-ends of RNA molecules. All rNTrs share a related active-site ...architecture first described for DNA polymerase beta and a catalytic mechanism conserved among DNA and RNA polymerases. The best known examples are the nuclear poly(A) polymerases involved in the 3'-end processing of eukaryotic messenger RNA precursors and the ubiquitous CCA-adding enzymes that complete the 3'-ends of tRNA molecules. In recent years, a growing number of new enzymes have been added to the list that now includes the "noncanonical" poly(A) polymerases involved in RNA quality control or in the readenylation of dormant messenger RNAs in the cytoplasm. Other members of the group are terminal uridylyl transferases adding single or multiple UMP residues in RNA-editing reactions or upon the maturation of small RNAs and poly(U) polymerases, the substrates of which are still not known. 2'-5'Oligo(A) synthetases differ from the other rNTrs by synthesizing oligonucleotides with 2'-5'-phosphodiester bonds de novo.
The structure determination of major allergens is a prerequisite for analyzing surface exposed areas of the allergen and for mapping conformational epitopes. These may be determined by experimental ...methods including crystallographic and NMR-based approaches or predicted by computational methods. In this review we summarize the existing structural information on allergens and their classification in protein fold families. The currently available allergen-antibody complexes are described and the experimentally obtained epitopes compared. Furthermore we discuss established methods for linear and conformational epitope mapping, putting special emphasis on a recently developed approach, which uses the structural similarity of proteins in combination with the experimental cross-reactivity data for epitope prediction.
Through alternative polyadenylation, human mRNAs acquire longer or shorter 3′ untranslated regions, the latter typically associated with higher transcript stability and increased protein production. ...To understand the dynamics of polyadenylation site usage, we performed transcriptome-wide mapping of both binding sites of 3′ end processing factors CPSF-160, CPSF-100, CPSF-73, CPSF-30, Fip1, CstF-64, CstF-64τ, CF Im25, CF Im59, and CF Im68 and 3′ end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF-64/CstF-64τ and CFIm proteins have much higher positional specificity compared to CPSF components. Knockdown of CF Im68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3′ end processing factor can modulate the length of 3′ untranslated regions across the transcriptome and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF Im68 knockdown.
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► Thousands of poly(A) sites are identified by the A-seq method in a human cell line ► Binding sites of pre-mRNA 3′ end processing factors are mapped by PAR-CLIP ► CstF-64 and CF Im68 exhibit the highest positional binding specificity ► CF Im 68 siRNA treatment causes a global shift toward proximal poly(A) sites
Alternative cleavage and polyadenylation generate mRNAs with 3′ untranslated regions of different lengths. Keller, Zavolan, and colleagues mapped the binding sites of ten 3′ end processing proteins by PAR-CLIP and identified the 3′ end processing sites by A-seq. CstF-64 and CF Im68 proteins showed the highest positional specificity. Knockdown of CF Im68 induced a systematic shift toward proximal polyadenylation sites, indicating that changes in the relative abundance of a single 3′ end processing factor can modulate the length of 3′ untranslated regions transcriptome-wide.
Type IV Secretion Systems (T4SSs) are membrane-spanning multiprotein complexes dedicated to protein secretion or conjugative DNA transport (conjugation systems) in bacteria. The prototype and ...best-characterized T4SS is that of the Gram-negative soil bacterium
. For Gram-positive bacteria, only conjugative T4SSs have been characterized in some biochemical, structural, and mechanistic details. These conjugation systems are predominantly encoded by self-transmissible plasmids but are also increasingly detected on integrative and conjugative elements (ICEs) and transposons. Here, we report regulatory details of conjugation systems from
model plasmids pIP501 and pCF10,
plasmid pLS1,
plasmid pCW3, and staphylococcal plasmid pSK41. In addition, regulation of conjugative processes of ICEs (ICE
1, ICE
1, ICE
3) by master regulators belonging to diverse repressor families will be discussed. A special focus of this review lies on the comparison of regulatory mechanisms executed by proteins belonging to the RRNPP family. These regulators share a common fold and govern several essential bacterial processes, including conjugative transfer.
Alternative polyadenylation (APA) is a general mechanism of transcript diversification in mammals, which has been recently linked to proliferative states and cancer. Different 3' untranslated region ...(3' UTR) isoforms interact with different RNA-binding proteins (RBPs), which modify the stability, translation, and subcellular localization of the corresponding transcripts. Although the heterogeneity of pre-mRNA 3' end processing has been established with high-throughput approaches, the mechanisms that underlie systematic changes in 3' UTR lengths remain to be characterized. Through a uniform analysis of a large number of 3' end sequencing data sets, we have uncovered 18 signals, six of which are novel, whose positioning with respect to pre-mRNA cleavage sites indicates a role in pre-mRNA 3' end processing in both mouse and human. With 3' end sequencing we have demonstrated that the heterogeneous ribonucleoprotein C (HNRNPC), which binds the poly(U) motif whose frequency also peaks in the vicinity of polyadenylation (poly(A)) sites, has a genome-wide effect on poly(A) site usage. HNRNPC-regulated 3' UTRs are enriched in ELAV-like RBP 1 (ELAVL1) binding sites and include those of the CD47 gene, which participate in the recently discovered mechanism of 3' UTR-dependent protein localization (UDPL). Our study thus establishes an up-to-date, high-confidence catalog of 3' end processing sites and poly(A) signals, and it uncovers an important role of HNRNPC in regulating 3' end processing. It further suggests that U-rich elements mediate interactions with multiple RBPs that regulate different stages in a transcript's life cycle.
Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal ...AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.
Eukaryotic pre-mRNAs are capped at their 5′ ends, polyadenylated at their 3′ ends, and spliced before being exported from the nucleus to the cytoplasm. Although the three processing reactions can be ...studied separately in vitro, they are coupled in vivo. We identified subunits of the U2 snRNP in highly purified CPSF and showed that the two complexes physically interact. We therefore tested whether this interaction contributes to the coupling of 3′ end processing and splicing. We found that CPSF is necessary for efficient splicing activity in coupled assays and that mutations in the pre-mRNA binding site of the U2 snRNP resulted in impaired splicing and in much reduced cleavage efficiency. Moreover, we showed that efficient cleavage required the presence of the U2 snRNA in coupled assays. We therefore propose that the interaction between CPSF and the U2 snRNP contributes to the coupling of splicing and 3′ end formation.
Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast ...protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNAMet (tRNAiMet). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNAiMet with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNAiMet by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background The low-affinity receptor for IgE, FcεRII (CD23), contributes to allergic inflammation through allergen presentation to T cells, regulation of IgE responses, and enhancement of ...transepithelial allergen migration. Objective We sought to investigate the interaction between CD23, chimeric monoclonal human IgE, and the corresponding birch pollen allergen Bet v 1 at a molecular level. Methods We expressed 4 CD23 variants. One variant comprised the full extracellular portion of CD23, including the stalk and head domain; 1 variant was identical with the first, except for an amino acid exchange in the stalk region abolishing the N-linked glycosylation site; and 2 variants represented the head domain, 1 complete and 1 truncated. The 4 CD23 variants were purified as monomeric and structurally folded proteins, as demonstrated by gel filtration and circular dichroism. By using a human IgE mAb, the corresponding allergen Bet v 1, and a panel of antibodies specific for peptides spanning the CD23 surface, both binding and inhibition assays and negative stain electron microscopy were performed. Results A hitherto unknown IgE-binding site was mapped on the stalk region of CD23, and the non–N-glycosylated monomeric version of CD23 was superior in IgE binding compared with glycosylated CD23. Furthermore, we demonstrated that a therapeutic anti-IgE antibody, omalizumab, which inhibits IgE binding to FcεRI, also inhibited IgE binding to CD23. Conclusion Our results provide a new model for the CD23-IgE interaction. We show that the stalk region of CD23 is crucially involved in IgE binding and that the interaction can be blocked by the therapeutic anti-IgE antibody omalizumab.
More than 10% of the world's population suffers from an immunoglobulin E (IgE)-mediated allergy to cats which is accompanied mainly by respiratory symptoms such as rhinitis and asthma. Several cat ...allergen molecules have been identified, but their allergenic activity has not been investigated in depth. Purified cat allergen molecules (Fel d 1, Fel d 2, Fel d 3, Fel d 4, Fel d 6, Fel d 7 and Fel d 8) were characterized via mass spectrometry and circular dichroism spectroscopy regarding their molecular mass and fold, respectively. Cat-allergen-specific IgE levels were quantified via ImmunoCAP measurements in IgE-sensitized subjects with (n = 37) and without (n = 20) respiratory symptoms related to cat exposure. The allergenic activity of the cat allergens was investigated by loading patients' IgE onto rat basophils expressing the human FcεRI receptor and studying the ability of different allergen concentrations to induce β-hexosaminidase release. Purified and folded cat allergens with correct masses were obtained. Cat-allergen-specific IgE levels were much higher in patients with a respiratory allergy than in patients without a respiratory allergy. Fel d 1, Fel d 2, Fel d 4 and Fel d 7 bound the highest levels of specific IgE and already-induced basophil degranulation at hundred-fold-lower concentrations than the other allergens. Fel d 1, Fel d 4 and Fel d 7 were recognized by more than 65% of patients with a respiratory allergy, whereas Fel d 2 was recognized by only 30%. Therefore, in addition to the major cat allergen Fel d 1, Fel d 4 and Fel d 7 should also be considered to be important allergens for the diagnosis and specific immunotherapy of cat allergy.
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