Zsofia Palfi

Interactome of Two Diverse RNA Granules Links mRNA Localization to Translational Repression in Neurons

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.

SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity.

Sm core variation in spliceosomal small nuclear ribonucleoproteins from Trypanosoma brucei

Messenger RNA processing in trypanosomes by cis and trans splicing requires spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5, as well as the spliced leader (SL) RNP. As in other eukaryotes, these RNPs share a core structure of seven Sm polypeptides. Here, we report that the identity of the Sm protein constituents varies between spliceosomal snRNPs: specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNP‐specific Sm proteins, Sm15K and Sm16.5K. We present a model for the variant Sm core in the U2 snRNP, based on tandem affinity purification‐tagging and in vitro protein–protein interaction assays. Using in vitro reconstitutions with canonical and U2‐specific Sm cores, we show that the exchange of two Sm subunits determines discrimination between individual Sm sites. In sum, we have demonstrated that the heteroheptameric Sm core structure varies between spliceosomal snRNPs, and that modulation of the Sm core composition mediates the recognition of small nuclear RNA‐specific Sm sites.

The trans-spliceosomal U2 snRNP protein 40K of Trypanosoma brucei: cloning and analysis of functional domains reveals homology to a mammalian snRNP protein.

Through immunoscreening we have isolated a cDNA encoding the trans‐spliceosomal U2 snRNP‐specific 40 kDa protein of Trypanosoma brucei. The protein has a predicted molecular weight of 36.6 kDa and shows 31% amino acid identity with the human U2 snRNP A′ protein of 28.4 kDa. The homology between the trypanosome and human protein sequences is restricted to the N‐terminal half where they share a series of six leucine repeat motifs. Sequence alignment revealed three 40K‐specific regions: a C‐terminal extension and two insertions, one of which makes up a seventh leucine repeat. Bacterially expressed 40K protein efficiently bound RNA by itself in a nonspecific manner; this general RNA binding activity was located to a region in the C‐terminal half overlapping with the leucine repeat domain. U2 RNA‐specific interaction required the presence of other trypanosome proteins and depended upon the loop IV sequence of U2 RNA. Deletion analysis of the 40K protein demonstrated the leucine repeats, including the 40K‐specific, seventh repeat, to be essential for specific U2 RNP assembly, most likely through their role as an interface for protein‐protein interaction.

Analysis of RNA-protein complexes by oligonucleotide-targeted RNase H digestion.

Oligonucleotide-targeted RNase H protection assays are powerful means to analyze protein binding domains in ribonucleoprotein particles (RNPs). In such an assay, the RNA component of a RNP and, in an essential control reaction, the corresponding deproteinized RNA are targeted with an antisense DNA oligonucleotide and RNase H. If the oligonucleotide is able to anneal to the complementary sequence of the RNA, RNase H will cleave the RNA within the double-stranded DNA/RNA region. However, protein binding to a specific RNA sequence may prevent hybridization of the DNA oligonucleotide, thereby protecting the RNA molecule from endonucleolytic cleavage. An RNase H protection analysis can usually be carried out with crude cell extract and does not require further RNP purification. On the other hand, purified RNP fractions are preferable when a crude extract contains RNase activity or a heterogenous RNP population of a specific RNA. The cleavage pattern of RNase H digestion can be analyzed by Northern blotting or primer-extension assays. In addition, the investigation of RNP fragments, for example, by native gel electrophoresis, may reveal important structural information about a RNP. In this article, we describe procedures for RNP and RNA preparation, the oligonucleotide-targeted RNase H protection assay, and methods for the analysis of RNA and RNP cleavage products. As an example, we show oligonucleotide-targeted RNase H protection of the Trypanosoma brucei U1 small nuclear RNP.

Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing

ABSTRACT Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.

Special Sm core complex functions in assembly of the U2 small nuclear ribonucleoprotein of Trypanosoma brucei.

ABSTRACT The processing of polycistronic pre-mRNAs in trypanosomes requires the spliceosomal small ribonucleoprotein complexes (snRNPs) U1, U2, U4/U6, U5, and SL, each of which contains a core of seven Sm proteins. Recently we reported the first evidence for a core variation in spliceosomal snRNPs; specifically, in the trypanosome U2 snRNP, two of the canonical Sm proteins, SmB and SmD3, are replaced by two U2-specific Sm proteins, Sm15K and Sm16.5K. Here we identify the U2-specific, nuclear-localized U2B″ protein from Trypanosoma brucei. U2B″ interacts with a second U2 snRNP protein, U2-40K (U2A′), which in turn contacts the U2-specific Sm16.5K/15K subcomplex. Together they form a high-affinity, U2-specific binding complex. This trypanosome-specific assembly differs from the mammalian system and provides a functional role for the Sm core variation found in the trypanosomal U2 snRNP.