Zophonias O. Jonsson

Small RNAs with Imperfect Match to Endogenous mRNA Repress Translation IMPLICATIONS FOR OFF-TARGET ACTIVITY OF SMALL INHIBITORY RNA IN MAMMALIAN CELLS

A 21-base pair RNA duplex that perfectly matches an endogenous target mRNA selectively degrades the mRNA and suppresses gene expression in mammalian tissue culture cells. A single base mismatch with the target is believed to protect the mRNA from degradation, making this type of interference highly specific to the targeted gene. A short RNA with mismatches to a target sequence present in multiple copies in the 3′-untranslated region of an exogenously expressed gene can, however, silence it by translational repression. Here we report that a mismatched RNA, targeted to a single site in the coding sequence of an endogenous gene, can efficiently silence gene expression by repressing translation. The antisense strand of such a mismatched RNA requires a 5′-phosphate but not a 3′-hydroxyl group. G·U wobble base pairing is tolerated as a match for both RNA degradation and translation repression. Together, these findings suggest that a small inhibitory RNA duplex can suppress expression of off-target cellular proteins by RNA degradation or translation repression. Proper design of experimental small inhibitory RNAs or a search for targets of endogenous micro-RNAs must therefore take into account that these short RNAs can affect expression of cellular genes with as many as 3–4 base mismatches and additional G·U mismatches.

Human-Specific Transcriptional Regulation of CNS Development Genes by FOXP2

The signalling pathways controlling both the evolution and development of language in the human brain remain unknown. So far, the transcription factor FOXP2 (forkhead box P2) is the only gene implicated in Mendelian forms of human speech and language dysfunction. It has been proposed that the amino acid composition in the human variant of FOXP2 has undergone accelerated evolution, and this two-amino-acid change occurred around the time of language emergence in humans. However, this remains controversial, and whether the acquisition of these amino acids in human FOXP2 has any functional consequence in human neurons remains untested. Here we demonstrate that these two human-specific amino acids alter FOXP2 function by conferring differential transcriptional regulation in vitro. We extend these observations in vivo to human and chimpanzee brain, and use network analysis to identify novel relationships among the differentially expressed genes. These data provide experimental support for the functional relevance of changes in FOXP2 that occur on the human lineage, highlighting specific pathways with direct consequences for human brain development and disease in the central nervous system (CNS). Because FOXP2 has an important role in speech and language in humans, the identified targets may have a critical function in the development and evolution of language circuitry in humans.

A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage.

ABSTRACT Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control.

Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain‐connecting loop of PCNA and loops on one face of the trimer, close to the C‐termini, is involved in binding to all of the following proteins: DNA polymerase δ, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase δ/PCNA. We demonstrate that PCNA must be located below a 5′ flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C‐termini facing forwards, in the direction of DNA synthesis.

Proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with Tudor family members

In germ cells, Piwi proteins interact with a specific class of small noncoding RNAs, piwi-interacting RNAs (piRNAs). Together, these form a pathway that represses transposable elements, thus safeguarding germ cell genomes. Basic models describe the overall operation of piRNA pathways. However, the protein compositions of Piwi complexes, the critical protein-protein interactions that drive small RNA production and target recognition, and the precise molecular consequences of conserved localization to germline structures, call nuage, remains poorly understood. We purified the three murine Piwi family proteins, MILI, MIWI, and MIWI2, from mouse germ cells and characterized their interacting protein partners. Piwi proteins were found in complex with PRMT5/WDR77, an enzyme that dimethylates arginine residues. By immunoprecipitation with specific antibodies and by mass spectrometry, we found that Piwi proteins are arginine methylated at conserved positions in their N termini. These modifications are essential to direct complex formation with specific members of the Tudor protein family. Recognition of methylarginine marks by Tudor proteins can drive the localization of Piwi proteins to cytoplasmic foci in an artificial setting, supporting a role for this interaction in Piwi localization to nuage, a characteristic that correlates with proper operation of the piRNA pathway and transposon silencing in multiple organisms.

MMS19 Assembles Iron-Sulfur Proteins Required for DNA Metabolism and Genomic Integrity

MMS19 Joins the CIA Iron-sulfur (Fe-S) proteins play a critical role in cell metabolism and particularly in DNA repair and replication. Mutants in eukaryotic gene MMS19 are particularly sensitive to DNA damaging agents, suggesting that it is involved in DNA repair, but the mutations can also have other wide-ranging effects on the cell (see the Perspective by Gottschling). Now, Stehling et al. (p. 195, published online 7 June) and Gari et al. (p. 243, published online 7 June) show that in both yeast and humans, MMS19 functions as part of the cytosolic Fe-S protein assembly (CIA) machinery. The MMS19 is part of a specialized CIA targeting complex that plays a role late in cytosolic Fe-S protein assembly to direct Fe-S cluster transfer from the CIA scaffold complex to a subset of Fe-S proteins, including a number associated with DNA metabolism. A protein thought to be involved in DNA repair is, in fact, responsible for inserting iron-sulfur clusters into enzymes. Instability of the nuclear genome is a hallmark of cancer and aging. MMS19 protein has been linked to maintenance of genomic integrity, but the molecular basis of this connection is unknown. Here, we identify MMS19 as a member of the cytosolic iron-sulfur protein assembly (CIA) machinery. MMS19 functions as part of the CIA targeting complex that specifically interacts with and facilitates iron-sulfur cluster insertion into apoproteins involved in methionine biosynthesis, DNA replication, DNA repair, and telomere maintenance. MMS19 thus serves as an adapter between early-acting CIA components and a subset of cellular iron-sulfur proteins. The function of MMS19 in the maturation of crucial components of DNA metabolism may explain the sensitivity of MMS19 mutants to DNA damage and the presence of extended telomeres.

Damage-induced phosphorylation of Sld3 is important to block late origin firing

Origins of replication are activated throughout the S phase of the cell cycle such that some origins fire early and others fire late to ensure that each chromosome is completely replicated in a timely fashion. However, in response to DNA damage or replication fork stalling, eukaryotic cells block activation of unfired origins. Human cells derived from patients with ataxia telangiectasia are deficient in this process due to the lack of a functional ataxia telangiectasia mutated (ATM) kinase and elicit radioresistant DNA synthesis after γ-irradiation2. This effect is conserved in budding yeast, as yeast cells lacking the related kinase Mec1 (ATM and Rad3-related (ATR in humans)) also fail to inhibit DNA synthesis in the presence of DNA damage. This intra-S-phase checkpoint actively regulates DNA synthesis by inhibiting the firing of late replicating origins, and this inhibition requires both Mec1 and the downstream checkpoint kinase Rad53 (Chk2 in humans). However, the Rad53 substrate(s) whose phosphorylation is required to mediate this function has remained unknown. Here we show that the replication initiation protein Sld3 is phosphorylated by Rad53, and that this phosphorylation, along with phosphorylation of the Cdc7 kinase regulatory subunit Dbf4, blocks late origin firing in Saccharomyces cerevisiae. Upon exposure to DNA-damaging agents, cells expressing non-phosphorylatable alleles of SLD3 and DBF4 (SLD3-m25 and dbf4-m25, respectively) proceed through the S phase faster than wild-type cells by inappropriately firing late origins of replication. SLD3-m25 dbf4-m25 cells grow poorly in the presence of the replication inhibitor hydroxyurea and accumulate multiple Rad52 foci. Moreover, SLD3-m25 dbf4-m25 cells are delayed in recovering from transient blocks to replication and subsequently arrest at the DNA damage checkpoint. These data indicate that the intra-S-phase checkpoint functions to block late origin firing in adverse conditions to prevent genomic instability and maximize cell survival.

A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation.

Proliferating cell nuclear antigen is best known as a DNA polymerase accessory protein but has more recently also been shown to have different functions in important cellular processes such as DNA replication, DNA repair, and cell cycle control. PCNA has been found in quaternary complexes with the cyclin kinase inhibitor p21 and several pairs of cyclin-dependent protein kinases and their regulatory partner, the cyclins. Here we show a direct interaction between PCNA and Cdk2. This interaction involves the regions of the PCNA trimer close to the C termini. We found that PCNA and Cdk2 form a complex together with cyclin A. This ternary PCNA-Cdk2-cyclin A complex was able to phosphorylate the PCNA binding region of the large subunit of replication factor C as well as DNA ligase I. Furthermore, PCNA appears to be a connector between Cdk2 and DNA ligase I and to stimulate phosphorylation of DNA ligase I. Based on our results, we propose the model that PCNA brings Cdk2 to proteins involved in DNA replication and possibly might act as an “adaptor” for Cdk2-cyclin A to PCNA-binding DNA replication proteins.

Independent Analysis of the Flagellum Surface and Matrix Proteomes Provides Insight into Flagellum Signaling in Mammalian-infectious Trypanosoma brucei

The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for host-parasite signaling.

Replication Factor C Interacts with the C-terminal Side of Proliferating Cell Nuclear Antigen

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases δ and ε. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hübscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433). Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution.