https://www.cell.com/cell/pdf/S0092-8674(09)00083-X.pdf

Origins and Mechanisms of miRNAs and siRNAs

MicroRNAs (miRNAs) are a large family of post-transcriptional regulators of gene expression that are ~21 nucleotides in length and control many developmental and cellular processes in eukaryotic organisms. Research during the past decade has identified major factors participating in miRNA biogenesis and has established basic principles of miRNA function. More recently, it has become apparent that miRNA regulators themselves are subject to sophisticated control. Many reports over the past few years have reported the regulation of miRNA metabolism and function by a range of mechanisms involving numerous protein-protein and protein-RNA interactions. Such regulation has an important role in the context-specific functions of miRNAs.

http://m.docente.unife.it/franco.pagani/lezione-1101a-mirna/articoli-smallrna/2010%20FIlipowic%20The%20widespread%20regulation%20of%20miRNA%20%20copy.pdf

The widespread regulation of microRNA biogenesis, function and decay.

MicroRNAs (miRNAs) are small noncoding RNAs that extensively regulate gene expression in animals, plants, and protozoa. miRNAs function posttranscriptionally by usually base-pairing to the mRNA 3′-untranslated regions to repress protein synthesis by mechanisms that are not fully understood. In this review, we describe principles of miRNA-mRNA interactions and proteins that interact with miRNAs and function in miRNA-mediated repression. We discuss the multiple, often contradictory, mechanisms that miRNAs have been reported to use, which cause translational repression and mRNA decay. We also address the issue of cellular localization of miRNA-mediated events and a role for RNA-binding proteins in activation or relief of miRNA repression.

Regulation of mRNA Translation and Stability by microRNAs

MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as development and cancer. Although their mode of action has attracted great attention, the principles governing their expression and activity are only beginning to emerge. Recent studies have introduced a paradigm shift in our understanding of the microRNA biogenesis pathway, which was previously believed to be universal to all microRNAs. Maturation steps specific to individual microRNAs have been uncovered, and these offer a plethora of regulatory options after transcription with multiple proteins affecting microRNA processing efficiency. Here we review the recent advances in knowledge of the microRNA biosynthesis pathways and discuss their impact on post-transcriptional microRNA regulation during tumour development.

/pdf/many-roads-to-maturity-microrna-biogenesis-pathways-and-xwvxbe5xac.pdf

Many roads to maturity: microRNA biogenesis pathways and their regulation

The Spliceosome: Design Principles of a Dynamic RNP Machine

Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome's active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of added factors. Comparisons of the composition of the precatalytic versus the catalytic spliceosome revealed a marked exchange of proteins during the transition from the B to the C complex, with apparent stabilization of Prp19-CDC5 complex proteins and destabilization of SF3a/b proteins. Disruption of purified C complexes led to the isolation of a salt-stable ribonucleoprotein (RNP) core that contained both splicing intermediates and U2, U5 and U6 small nuclear RNA plus predominantly U5 and human Prp19-CDC5 proteins and Prp19-related factors. Our data provide insights into the spliceosome's catalytic RNP domain and indicate a central role for the aforementioned proteins in sustaining its catalytically active structure.

/pdf/isolation-of-an-active-step-i-spliceosome-and-composition-of-59ilu07812.pdf

Isolation of an active step I spliceosome and composition of its RNP core.

Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45S activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35S snRNP particle.

/pdf/small-nuclear-ribonucleoprotein-remodeling-during-catalytic-1nh4yp9hd6.pdf

Small Nuclear Ribonucleoprotein Remodeling During Catalytic Activation of the Spliceosome

https://pure.mpg.de/pubman/item/item_588184_3/component/file_588183/426972.pdf

Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.

Detailed knowledge of the composition and structure of the spliceosome and its assembly intermediates is a prerequisite for understanding the complex process of pre-mRNA splicing. To this end, we have developed a tobramycin affinity-selection method that is generally applicable for the purification of native RNP complexes. By using this method, we have isolated human prespliceosomes that are ideally suited for both biochemical and structural studies. MS identified >70 prespliceosome-associated proteins, including nearly all known U1 and U2 snRNP proteins, and expected non-snRNP splicing factors. In addition, the DEAD-box protein p68, RNA helicase A, and a number of proteins that appear to perform multiple functions in the cell, such as YB-1 and TLS, were detected. Several previously uncharacterized proteins of unknown function were also identified, suggesting that they play a role in splicing and potentially act during prespliceosome assembly. These data provide insight into the complexity of the splicing machinery at an early stage of its assembly.

https://www.pnas.org/content/pnas/99/26/16719.full.pdf

Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method.

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis. Pre-mRNA splicing is catalyzed by a large RNP molecular machine, termed the spliceosome, which consists of the U1, U2, U4/U6, and U5 snRNPs and a multitude of non-snRNP proteins (reviewed in reference 48). The active site(s) responsible for the catalysis of pre-mRNA splicing is not preformed but, rather, is created anew during the highly dynamic process of spliceosome assembly. The latter is an ordered process during which several intermediates, termed E, A, B, and B*, can be detected in vitro (reviewed in reference 48). Assembly is initiated by the ATP-independent interaction of the U1 snRNP with the conserved 5 splice site of the pre-mRNA, forming the E complex. At this stage, the U2 snRNP is loosely associated with the pre-mRNA (11). In a subsequent step requiring ATP, the U2 snRNP stably interacts with the pre-mRNA’s branch site, leading to formation of the A complex (also called the prespliceosome). Spliceosome assembly culminates with the formation of the spliceosomal B complex, during which the preformed U4/U6.U5 tri-snRNP particle interacts with the A complex. The B complex thus contains a full set of U snRNAs in a precatalytic state. It subsequently undergoes major rearrangements, including destabilization or loss of the U1 and U4 snRNPs, leading to catalytic activation and the formation of the so-called activated spliceosome (B* complex).

/pdf/protein-composition-and-electron-microscopy-structure-of-4ycgglrcef.pdf

Henning Urlaub

Papers

Isolation of an active step I spliceosome and composition of its RNP core.

Small Nuclear Ribonucleoprotein Remodeling During Catalytic Activation of the Spliceosome

Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.

Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method.

Protein Composition and Electron Microscopy Structure of Affinity-Purified Human Spliceosomal B Complexes Isolated under Physiological Conditions