From cis-regulatory elements to complex RNPs and back

Abstract

Messenger RNAs (mRNAs), the templates for translation, have evolved to harbor abundant cis-acting sequences that affect their posttranscriptional fates. These elements are frequently located in the untranslated regions and serve as binding sites for trans-acting factors, RNA-binding proteins, and/or small non-coding RNAs. This article provides a systematic synopsis of cis-acting elements, trans-acting factors, and the mechanisms by which they affect translation. It also highlights recent technical advances that have ushered in the era of transcriptome-wide studies of the ribonucleoprotein complexes formed by mRNAs and their trans-acting factors.

Figure 1.

Cis-acting elements that influence mRNA translation. The nearly ubiquitous 5′ ^m7GpppN cap structure (black circle) and 3′ poly(A) tail ((A)_n) strongly stimulate translation. Secondary structures (e.g., hairpin) and upstream open reading frames (uORFs) in the 5′ UTR usually inhibit translation. Internal ribosome entry sequences (IRES) stimulate translation independently of the cap structure. Binding sites for regulatory RNA-binding proteins or microRNAs (ovals) can provide positive or (more frequently) negative regulation. (Adapted and modified from Gebauer and Hentze 2004.)

Figure 2.

Methods to study mRNP composition. (A) Photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP). Cells are cultured in media containing 4-thiouridine (4SU), leading to incorporation of the photoactivatable nucleoside into cellular RNA. Cross-linking with UV light of 365 nm leads to covalent attachment of RBPs to RNA targets that withstands partial RNAse digestion, immunoprecipitation, and purification by denaturing gel electrophoresis. Isolated RNA fragments are identified by next-generation sequencing, aided by a tendency of the cross-linked site to show thymidine (T) to cytidine (C) transitions. (B) GRNA chromatography using specific interaction between a 21-amino-acid peptide from the λ phage N anti-terminator protein and the boxB hairpin. A fusion of λN peptide with glutathione S-transferase (GST), and incorporation of the boxB hairpin into bait RNA converts glutathione Sepharose into an RNA affinity matrix (GRNA resin), which is incubated with cellular extracts. Proteins specifically bound to the matrix are eluted and identified by mass spectrometry. (C) Interactome capture. The procedure begins with RNP cross-linking in living cells by conventional UV 254-nm cross-linking or as in the PAR-CLIP approach. Following lysis, the complete cellular complement of (m)RNPs is purified by binding to an oligo(dT) resin and stringent washing under conditions that dissociate noncovalent RNA–protein interactions. Specifically bound proteins are released by RNase digestion and identified by mass spectrometry. (Diagrams are based on data from Czaplinski et al. 2005, Hafner et al. 2010, and Castello et al. 2012, respectively.)

Figure 3.

Mechanism of translational repression of msl2 mRNA. SXL binds to both the 5′ and 3′ UTRs of msl2 to achieve strong repression. SXL bound to the 3′ UTR recruits UNR to bind to the RNA in close proximity. In turn, UNR interacts with poly(A) tail-bound PABP to inhibit 43S ribosomal complex recruitment at a step downstream from closed-loop formation (1). SXL bound to the 5′ UTR inhibits ribosomal scanning by promoting recognition of an upstream AUG (uAUG), thus preventing 43S complexes from reaching the main msl2 ORF (2). Additional unidentified factors (X, Y) are likely involved. (Adapted in modified form from Graindorge et al. 2011.)

Figure 4.

Mechanism of translational repression of nanos mRNA. nanos mRNA switches from a translationally active state in nurse cells to a silenced state in late oocytes and early embryos. Repression in late oocytes is driven by Glorund (Glo) binding to stem IIIA of the TCE and seems to be effected primarily at the elongation step. In embryos, Smaug (Smg) is synthesized and takes over repression by binding to the SREs within the TCE; the relative contribution of each SRE is uncertain, although SRE1 seems to contribute more to Smaug binding than SRE2. Smg recruits the eIF4E-binding protein Cup and the CAF–CCR4–NOT complex to block translation initiation and promote deadenylation and degradation of nos mRNA. The piwi pathway has been recently reported to participate in deadenylation. Additional steps of translation could be affected both in late oocytes and early embryos (see text for details).

Figure 5.

Translational repression by the GAIT complex. During inflammation, EPRS and L13a are phosphorylated and released from the multisynthetase complex (MSC) and the 60S ribosomal subunit, respectively. These proteins bind to NSAP1 and GAPDH to form the heterotetrameric GAIT complex, which binds to a split stem structure present in the 3′ UTR of target mRNAs. L13a then inhibits the recruitment of the 43S ribosomal complex by blocking the interaction between 43S-associated eIF3 and eIF4G.