During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.
The cell wall of a diatom is made up of a silica-based scaffold and organic macromolecules. Proteins located in the cell wall are believed to control morphogenesis of the species-specific silica structures of the scaffold. However, data that correlate distinct silica elements and specific proteins within the diatom cell wall have not been reported. Here, the cell wall protein HEP200 (200-kDa HF-extractable protein) from the diatom Cylindrotheca fusiforrnis is identified and characterized. HEP200 is tightly associated with a substructure of the silica scaffold. It is a member of a new protein family, of which two more members are identified. Each member displays the same bipartite structure. The N-terminal part consists of a variable number of a repeated sequence motif (PSCD domain), whereas the C-terminal part is unique. Immunolocalization experiments revealed the arrangement of different proteins within the cell wall. Frustulins, a previously described group of glycoproteins, constitute the outer coat of the cell wall and exhibit a ubiquitous distribution. In contrast, HEP200 is specifically located at a subset of about six silica strips in intact cell walls, shielded by frustulins. This study therefore identifies a diatom cell wall protein (HEP200) that is associated with a distinct substructure of the silica scaffold.Keywords: biomineralization ; Cylinclrotheca fusiforrnis; girdle band; immunolocalization ; morphogenesis.Diatoms are unicellular organisms that constitute the largest class among the algae (van den Hoek et al., 1993). One of the most fascinating aspects of diatom biology is the species-specific design and ornamentation of the cell wall. The scaffold of diatom cell walls is made of inorganic material, namely amorphous, hydrated siliciumdioxid (silica), which is associated with proteins and polysaccharides (Volcani, 1981). It is the structure and arrangement of the silica scaffold that gives rise to the intricate patterns of the cell wall. A similar cell wall composition is also found within the Synurophyceae (McGrory and Leadbeater, 1981).To create their silica-based cell walls, these organisms have developed a common silica secretion mechanism, which has been intensively studied in diatoms (Pickett-Heaps et a]., 1990). Soluble silicate is taken up from the environment and concentrated in a special, intracellular organelle, the silica-deposition vesicle (SDV). Within the SDV, silicate polymerizes yielding insoluble silica. It is at this stage of cell wall biogenesis that species-specific patterning of the silica scaffold takes place. Subsequently, the SDV membrane (silicalemma) and plasmamembrane fuse, which deposits the silica scaffold outside the cell. It is not clear, whether diatom cell wall proteins and polysacchaCorrespondence to N. Kroger,
Pattern formation during silica biomineralization in diatoms appears to depend on long-chain polyamines as well as proteins covalently modified with polyamines (silaffins). Recently, the complete genome of the diatom Thalassiosira pseudonana has been sequenced making this species an attractive model organism for future studies on biomineralization. Mass-and NMR-spectroscopic analysis of the long-chain polyamines from this diatom species reveals the existence of a complex population with as yet unknown structural features. These include complex methylation patterns, different attachment moieties as well as the existence of quaternary ammonium functionalities.
The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading.
Diatoms are eukaryotic, unicellular algae that are well known for the intricate architecture of their silica-based cell walls. Species identification is mainly based on variations of their hierarchically organized silica structures. Particularly striking silica frameworks are found among diatoms that belong to the genus Coscinodiscus. Recent work indicates an important role for long-chain polyamines in guiding silica precipitation as well as in silica-pattern formation. Here we demonstrate that polyamines, even if isolated from closely related diatom species, exhibit substantial structural differences. Structural variations include the overall chain length, the degree of methylation, positions of secondary amino functionalities, and, unexpectedly, site-specific incorporation of a quaternary ammonium functionality. These findings support a specific role for polyamines in creating silica nanostructures.
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