Nghiên cứu mới về vai trò của protein thách thức nhận thức xưa nay trong sách giáo khoa

Posted on January 14, 2015 by

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Jan 1, 2015 (Phys.org) — Mở sách giáo khoa sinh học nào thì một trong những bài học đầu tiên là vai trò của DNA chỉ dẫn/ra lệnh quá trình sản xuất các protein. Proteins là những “cỗ máy” tí hon đảm nhận vô số chức năng và công việc trong các tế bào của cơ thể chúng ta.

Kết quả nghiên cứu công bố trong bài báo dưới đây trên Science ngày 2-1-2015 đã thách thức nhận thức xưa nay trong sách giáo khoa như vừa nêu ở trên. Lần đầu tiên, các nhà nghiên cứu chỉ ra rằng những yếu tố cơ bản làm nên các protein – gọi là amino acids – có thể được sản xuất mà không cần các lệnh chỉ dẫn của DNA và một sơ đồ khuôn mẫu trung gian gọi là messenger RNA (mRNA). Nhóm tác giả đã quan sát được trường hợp trong đó một protein khác đã chỉ thị bổ sung những amino acids cần bổ sung.

“Phát hiện gây kinh ngạc này phản ánh hiểu biết sinh học của chúng ta khiếm khuyết tới mức nào,” tác giả dẫn đầu Peter Shen, Ph.D., tại University of Utah nói. “Thiên nhiên cho thấy năng lực lớn hơn nhiều so với những gì ta biết.”

A new finding goes against dogma, showing for the first time that the building blocks of a protein, called amino acids, can be assembled by another protein, and without genetic instructions). The Rqc2 protein (yellow) binds tRNAs (dark blue, teal) which add amino acids (bright spot in middle) to a partially made protein (green). The complex binds the ribosome (white). Credit: Janet Iwasa, Ph.D., University of Utah

A new finding goes against dogma, showing for the first time that the building blocks of a protein, called amino acids, can be assembled by another protein, and without genetic instructions). The Rqc2 protein (yellow) binds tRNAs (dark blue, teal) which add amino acids (bright spot in middle) to a partially made protein (green). The complex binds the ribosome (white). Credit: Janet Iwasa, Ph.D., University of Utah

Để hình dung kết quả này, có thể coi tế bào nhưng một nhà máy được vận hành trơn tru. Ribosomes là những cỗ máy trên dây chuyền lắp ráp protein, liên kết các theo một trật tự được chỉ thị bởi mã gien. Nếu có gì đó trục trặc, ribosome có thể ngưng hoạt động, và một đội kiểm tra chất lượng được triệu tập đến điểm “sự cố”. Để giải quyết lộn xộn, ribosome sẽ được tháo dỡ, sơ đồ chỉ dẫn sản xuất bị dẹp bỏ, và protein đang sản xuất dở dang sẽ phải tái chế.

Song nghiên cứu  này chỉ ra một vai trò đáng ngạc nhiên của một thành viên của đội kiểm tra chất lượng, một protein có tên gọi Rqc2. Trước khi protein sản xuất dở dang được tái chế, Rqc2 lập tức yêu cầu ribosomes bổ sung hai amino acids (trên tổng số 20) – alanine and threonine – liên tục, và theo bất kỳ trật tự cần thực hiện. Hãy coi đó như một dây chuyền lắp xe hơi vẫn tiếp tục chạy ngay cả khi bị mất hướng dẫn quy trình sản xuất. Nó nhặt loại vật tư nào có thể và lắp theo trình tự nào đó, ví dụ: còi-bánh-bánh-còi-bánh-bánh-bánh-bánh-còi.

“Trong trường hợp này, ta có một protein đóng vai trò thường do mRNA đảm nhiệm,” theo Adam Frost, Ph.D., tại University of California, San Francisco (UCSF). “Tôi rất thích câu chuyện này vì xóa mờ đường biên nhận thức chúng ta nghĩ về khả năng của các protein.”

Giống như một chiếc xe hơi đang lắp dở và có thừa còi và bánh, nên lắp vào một đầu, một protein bị rút ngắn với một chuỗi ngẫu nhiên các alanines và threonines trông kỳ dị, và có khả năng không vận hành bình thường được. Nhưng một chuỗi trông vô lý lại có thể phục vụ một vài mục tiêu rất cụ thể. Mã đó có thể gửi tín hiệu nhận biết rằng protein dở dang đó cần phải bị phá hủy, hoặc nó có thể trở thành một phần của phép kiểm tra xem liệu bản thân ribosome có hoạt động hợp lý. Bằng chứng trên nói rằng một hoặc cả hai quá trình này có thể bị lỗi trong những căn bệnh suy thoái thần kinh như Alzheimer’s, Amyotrophic lateral sclerosis (ALS), hoặc Huntington.

Khi thấy hiện tượng bất thường qua quan sát bằng mắt, các nhà khoa học đã tinh chỉnh một kỹ thuật gọi là “cryo-electron microscopy” để dừng nhanh và quan sát, cỗ máy kiểm soát chất lượng trong quá trình vận hành. “Chúng tôi bắt quả tang Rqc2 tại trận,” theo tác giả Frost. “Nhưng nhiệm vụ lớn hơn nhiều việc quan sát thuần túy. Chúng tôi phải chứng minh quá trình đó.”

Rất nhiều phân tích hóa sinh được tiến hành nhằm chứng minh giả thiết. Các kỹ thuật sequencing RNA chỉ ra rằng tổ hợp Rqc2/ribosome có tiềm năng bổ sung amino acids vào các protein đang bị dừng sản xuất vì nó cũng nối các tRNAs, là những cấu trúc vận chuyển amino acids với các dây chuyền sản xuất . Các tRNAs cụ thể được quan sát trực tiếp trong nghiên cứu này chỉ vận chuyển hai amino acids là alanine và threonine. Lập luận quyết định được đưa ra khi họ xác định được rằng những proteins bị ngưng sản xuất chứa những chuỗi alanines và threonines được bổ sung thêm bất thường.

“Tiếp theo nhiệm vụ của chúng tôi là phải xác định vào lúc nào và ở chỗ nào quá trình này xảy ra, và điều gì xảy đến nếu quá trình này thất bại,” tác giả Frost phát biểu.

More information: Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Peter S. Shen, Joseph Park, Yidan Qin, Xueming Li, Krishna Parsawar, Matthew H. Larson, James Cox, Yifan Cheng, Alan M. Lambowitz, Jonathan S. Weissman, Onn Brandman, Adam Frost. Science, Jan. 2, 2015. www.sciencemag.org/lookup/doi/… 1126/science.1259724

Dưới đây là bài nghiên cứu trên tạp chí Science:

Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

Despite the processivity of protein synthesis, faulty messages or defective ribosomes can result in translational stalling and incomplete nascent chains. In Eukarya, this leads to recruitment of the ribosome quality control complex (RQC), which mediates the ubiquitylation and degradation of incompletely synthesized nascent chains (14). The molecular components of the RQC include the AAA adenosine triphosphatase Cdc48p and its ubiquitin-binding cofactors, the RING-domain E3 ligase Ltn1p, and two proteins of unknown function, Rqc1p and Rqc2p. We set out to determine the mechanism(s) by which relatively rare (5) proteins such as Ltn1p, Rqc1p, and Rqc2p recognize and rescue stalled 60S ribosome nascent chain complexes, which are vastly outnumbered by ribosomes translating normally or in stages of assembly.

To reduce structural heterogeneity and enrich for complexes still occupied by stalled nascent chains, we immunoprecipitated Rqc1p-bound RQC assemblies from Saccharomyces cerevisiae strains lacking the C-terminal RING domain of Ltn1p, which prevents substrate ubiquitylation and Cdc48 recruitment (1). Three-dimensional (3D) classification of Ltn1ΔRING particles revealed 60S ribosomes with nascent chains in the exit tunnel and extraribosomal densities (Fig. 1). These extraribosomal features were resolved between 5 and 14Å and proved to be either Tif6p or RQC components as characterized below (Fig. 1 and figs. S1 to S7). Tif6p was not observed bound to the same 60S particles bound by RQC factors (figs. S1 to S3). We repeated the purification, imaging, and 3D classification from rqc2Δ cells and computed difference maps. This analysis did not reveal density attributable to Rqc1p but did identify Rqc2p as a transfer RNA (tRNA)–binding protein that occupies the 40S binding surface and Ltn1p as the elongated molecule that meets Rqc2p at the sarcin-ricin loop (SRL) (Figs. 1 and 2 and figs. S1 to S5). Comparison of the 60S-bound Ltn1p with reconstructions of isolated Ltn1p suggests that the N terminus of Ltn1p engages the SRL with Rqc2p and that the middle region—which contains long HEAT/Armadillo repeats that adopt an elongated superhelical structure—reaches around the 60S (6). This conformation probably positions the C-terminal RING domain near the exit tunnel to ubiquitylate stalled nascent chains [figs. S5 and S6 and (7)].

Fig. 1 Cryo-EM reconstructions of peptidyl-tRNA-60S ribosomes bound by the RQC components Rqc2p and Ltn1p. (A) A peptidyl-tRNA-60S complex isolated by immunoprecipitation of Rqc1p. The ribosome density is transparent to visualize the nascent chain. (B) Rqc2p (purple) and an ~A-site tRNA (yellow) bound to peptidyl-tRNA-60S complexes. Landmarks are indicated (L1, L1 stalk; SB, P-stalk base). (C) Ltn1p (tan) bound to Rqc2p-peptidyl-tRNA-60S complexes (B).

Fig. 1 Cryo-EM reconstructions of peptidyl-tRNA-60S ribosomes bound by the RQC components Rqc2p and Ltn1p.
(A) A peptidyl-tRNA-60S complex isolated by immunoprecipitation of Rqc1p. The ribosome density is transparent to visualize the nascent chain. (B) Rqc2p (purple) and an ~A-site tRNA (yellow) bound to peptidyl-tRNA-60S complexes. Landmarks are indicated (L1, L1 stalk; SB, P-stalk base). (C) Ltn1p (tan) bound to Rqc2p-peptidyl-tRNA-60S complexes (B).

Fig. 2 Rqc2p binding to the 60S ribosome and ~P-site, and ~A-site tRNAs. (A) Rqc2p contacts ~P- and ~A-site tRNAs, the SRL, and P-stalk base ribosomal RNA (SB). (B) Rigid body fitting of tRNAs structures (ribbons) into EM densities (mesh).

Fig. 2 Rqc2p binding to the 60S ribosome and ~P-site, and ~A-site tRNAs.
(A) Rqc2p contacts ~P- and ~A-site tRNAs, the SRL, and P-stalk base ribosomal RNA (SB). (B) Rigid body fitting of tRNAs structures (ribbons) into EM densities (mesh).

A refined reconstruction of the Rqc2p-occupied class demonstrated that Rqc2p makes extensive contacts with an approximately P-site positioned (~P-site) tRNA (Figs. 1 and 2 and fig. S7). Rqc2p has a long coiled coil that makes direct contact with the SRL and the 60S P-stalk base (Fig. 2A). This structure also revealed Rqc2p binding to an ~A-site tRNA, whose 3′-CCA tail is within the peptidyl transferase center of the 60S (Fig. 2B and fig. S7). This observation was unexpected, because A-site tRNA interactions with the large ribosomal subunit are typically unstable and require mRNA templates and elongation factors (8). Rqc2p’s interactions with the ~A-site tRNA appeared to involve recognition between the anticodon loop and a globular N-terminal domain, as well as D-loop and T-loop interactions along Rqc2p’s coiled coil (Figs. 2 and 3).

Fig. 3 Rqc2p-dependent enrichment of tRNAAla(IGC) and tRNAThr(IGU). (A) tRNA cDNA reads extracted from purified RQC particles and summed per unique anticodon, with versus without Rqc2p. (B) Secondary structures of tRNAAla(IGC) and tRNAThr(IGU). Identical nucleotides are underlined. Edited nucleotides are indicated with asterisks (24, 25). (C) Weblogo representation of cDNA sequencing reads related to shared sequences found in anticodon loops (positions 32 to 38) of mature tRNAAla(IGC) and tRNAThr(IGU) (26). (D) ~A-tRNA contacts with Rqc2p at the D-, T-, and anticodon loops. Identical nucleotides between tRNAAla(IGC) and tRNAThr(IGU) are colored as in (B) (A, green; U, red; C, blue; G, orange) and pyrimidine, purple. Anticodon nucleotides are indicated as slabs.

Fig. 3 Rqc2p-dependent enrichment of tRNAAla(IGC) and tRNAThr(IGU).
(A) tRNA cDNA reads extracted from purified RQC particles and summed per unique anticodon, with versus without Rqc2p. (B) Secondary structures of tRNAAla(IGC) and tRNAThr(IGU). Identical nucleotides are underlined. Edited nucleotides are indicated with asterisks (24, 25). (C) Weblogo representation of cDNA sequencing reads related to shared sequences found in anticodon loops (positions 32 to 38) of mature tRNAAla(IGC) and tRNAThr(IGU) (26). (D) ~A-tRNA contacts with Rqc2p at the D-, T-, and anticodon loops. Identical nucleotides between tRNAAla(IGC) and tRNAThr(IGU) are colored as in (B) (A, green; U, red; C, blue; G, orange) and pyrimidine, purple. Anticodon nucleotides are indicated as slabs.

To determine whether Rqc2p binds specific tRNA molecules, we extracted total RNA after RQC purification from strains with intact RQC2 versus rqc2Δ strains. Deep sequencing by a new method using a thermostable group II intron reverse transcriptase (9) revealed that the presence of Rqc2p leads to an ~10-fold enrichment of tRNAAla(AGC) and tRNAThr(AGT) in the RQC (Fig. 3A). In complexes isolated from strains with intact RQC2, Ala(AGC) and Thr(AGT) are the most abundant tRNA molecules, even though they are less abundant than a number of other tRNAs in yeast (10).

Our structure suggested that Rqc2p’s specificity for these tRNAs is due in part to direct interactions between Rqc2p and positions 32 to 36 of the anticodon loop, some of which are edited in the mature tRNA (Fig. 3). Adenosine 34 in the anticodon of both tRNAAla(AGC) and tRNAThr(AGT) is deaminated to inosine (1113), leading to a diagnostic guanosine upon reverse transcription (13, 14) (Fig. 3, B and C). Further analysis of the sequencing data revealed that cytosine 32 in tRNAThr(AGT) is also deaminated to uracil in ~70% of the Rqc2p-enriched reads [Fig. 3 and (15)]. Together with the structure, this suggests that Rqc2p binds to the D-, T-, and anticodon loop of the ~A-site tRNA, and that recognition of the 32-UUIGY-36 edited motif accounts for Rqc2’s specificity for these two tRNAs (Fig. 3, C and D). The pyrimidine at position 36 could explain the discrimination between the otherwise similar anticodon loops that harbor purines at base 36.

While assessing why Rqc2p evolved to bind these specific tRNA molecules, we considered these observations: First, our structural and biochemical data indicate that Rqc2p binds the 60S subunit after a stalled ribosome dissociates [fig. S6 (1, 2)]. Second, stalled nascent chains accumulate as higher-molecular-weight species in the presence of Rqc2p than in its absence [Fig. 4A, also seen in Fig. 3E of (1)]. Finally, amino acid addition to a nascent chain can be mediated by the large ribosomal subunit in vitro even when decoupled from an mRNA template and the small subunit (16). Together, these facts led us to hypothesize that Rqc2p may promote the extension of stalled nascent chains with alanine and threonine residues in an elongation reaction that is mRNA- and 40S-free. This hypothesis makes specific predictions. First, the Rqc2p-dependent increase in the molecular weight of the nascent chain should occur from the C terminus exclusively. Second, the C-terminal extension should consist entirely of alanine and threonine residues that start immediately at the stalling sequence. Finally, the alanine and threonine extension should not have a defined sequence.

Fig. 4 Rqc2p-dependent formation of CAT tails. (A, B, and D) Immunoblots of stalling reporters in RQC deletion strains. (C) Total amino acid analysis of immunoprecipitated GFP expressed in ltn1Δ and ltn1Δrqc2Δ strains, n = 3 independent immunoprecipitations. (E) Triplicate GFP levels measured with a flow cytometer and normalized to a wild-type control. EV, empty vector. All error bars are standard deviations.

Fig. 4 Rqc2p-dependent formation of CAT tails.
(A, B, and D) Immunoblots of stalling reporters in RQC deletion strains. (C) Total amino acid analysis of immunoprecipitated GFP expressed in ltn1Δ and ltn1Δrqc2Δ strains, n = 3 independent immunoprecipitations. (E) Triplicate GFP levels measured with a flow cytometer and normalized to a wild-type control. EV, empty vector. All error bars are standard deviations.

To test these predictions, we expressed a series of reporters containing a stalling sequence [tracts of up to 12 consecutive arginine codons, including pairs of the difficult-to-decode CGA codon (17)], inserted between the coding regions of green fluorescent protein (GFP) and red fluorescent protein (Fig. 4A). Null mutations in RQC components or inhibition of the proteasome led to the accumulation of nascent chain fragments that are normally degraded in wild-type cells (Fig. 4A) (14, 18). Furthermore, ltn1Δ and rqc2Δ cells have different phenotypes: Expression of the stalling reporter in ltn1Δ led to the formation and accumulation of higher-molecular-weight species that resolve as a smear ~1.5 to 5 kD above the expected position of GFP (Fig. 4A). GFP mass-shifted products are observable in rqc1Δltn1Δ double mutants, less prominent but still observable in rqc1Δ single mutants, but absent in all rqc2Δ single and double mutants (Fig. 4A). Thus, Rqc2p is necessary for the production of these higher-molecular-weight GFP species.

We probed the location of the extra mass along the GFP by inserting a tobacco etch virus (TEV) protease cleavage site upstream of the stalling tract (Fig. 4B). GFP resolved as a single band of the expected size with TEV treatment, indicating that the extra mass is located at or after the stall sequence. To pinpoint the location of the extra mass along the GFP, we moved the TEV cleavage site after the R12 stalling sequence. This created a mass-shifted GFP that was insensitive to TEV treatment, suggesting that the post-R12 TEV cleavage site was not synthesized. One possible model is that a translational frameshift occurs near the R12 sequence, which causes the mRNA to be mistranslated until the next out-of-frame stop codon. We falsified this model in two ways. First, we detected an Rqc2p-dependent GFP mass shift using a shorter R4 reporter in which multiple STOP codons were engineered in the +1 and +2 frames following the polyarginine tract (fig. S8). Second, we detected the Rqc2p-dependent GFP mass shift in a construct encoding a hammerhead ribozyme. The ribozyme cleaves the coding sequence of the GFP mRNA, leaving a truncated non-stop mRNA that causes a stall during translation of its final codon [fig. S8 (19)]. Thus, the GFP mass shift is located at or after the stall sequence but cannot be explained by mRNA translation past the stalling tract in any frame.

In order to determine the composition of the GFP mass-shifted products, we performed total amino acid analysis of immunopurified GFP from strains expressing the stalling reporter. Purified GFP from ltn1Δ (Fig. 4C) or rqc1Δ strains (fig. S9) is enriched in alanine and threonine as compared to purified GFP from double mutants with rqc2Δ which do not produce extended GFP. We then used Edman degradation to sequence TEV release fragments after purification of the stalled GFP reporter from the ltn1Δ strain. The first three codons in the R12 sequence are CGG-CGA-CGA, and Edman degradation suggested that the ribosome stalls at the first pair of the challenging-to-decode CGA codons (fig. S10). Following the encoded arginine residues, rising levels of alanine and threonine were detected at the C terminus (fig. S10). We further characterized these fragments by mass spectrometry and detected diverse poly-Ala and poly-Thr species ranging from 5 to 19 residues, with no defined sequence (table S1). Together, these observations demonstrate that Rqc2p directs the elongation of stalled nascent chains with nontemplated carboxy-terminal Ala and Thr extensions, or “CAT tails.”

Earlier work (1) revealed that the accumulation of stalled nascent chains (e.g., by deletion of LTN1) led to a robust heat shock response that is fully dependent on Rqc2p, although the mechanism by which Rqc2p enabled this stress response was unclear. We hypothesized that CAT tails may be required for activation of heat shock factor 1 (Hsf1p). To isolate the effect of CAT tails in this context, we sought an rqc2 allele that could not support CAT tail synthesis but could still bind to the 60S and facilitate Ltn1p-dependent ubiquitylation of the nascent chains. Rqc2p belongs to the conserved NFACT family of nucleic acid–binding proteins (20), and the N-terminal NFACT-N domain of Rqc2p is 22% identical to the NFACT-N domain of the Staphylococcus aureus protein Fbp (PDB:3DOA). Based on sequence and predicted secondary structure conservation, we fit this structure into a portion of the cryo–electron microscopy (cryo-EM) density ascribed to Rqc2p (figs. S11 and S12). This modeling exercise predicts that Rqc2p’s NFACT-N domain recognizes features of both the P- and A-site tRNA molecules and that conserved residues D9, D98, and R99, which have been hypothesized to play roles in nucleic acid–binding or –modifying reactions (20), may contact the ~A-site tRNA (20) (fig. S12). An Rqc2p variant in which these residues were mutated to alanine (rqc2aaa) rescued 60S recognition and the clearance of the stalling reporter almost as effectively as wild-type Rqc2p but did not support CAT tail synthesis (Fig. 4D and fig. S12). This CAT tail–deficient rqc2aaa allele also failed to rescue Hsf1p transcriptional activation (Fig. 4E), indicating that CAT tails may promote Hsf1p activation.

Integrating our observations, we propose the model schematized in fig. S13. Ribosome stalling leads to dissociation of the 60S and 40S subunits, followed by recognition of the peptidyl-tRNA-60S species by Rqc2p and Ltn1p. Ltn1p ubiquitylates the stalled nascent chain, and this leads to Cdc48 recruitment for extraction and degradation of the incomplete translation product. Rqc2p, through specific binding to Ala(IGC) and Thr(IGU) tRNAs, directs the template-free and 40S-free elongation of the incomplete translation product with CAT tails. CAT tails induce a heat shock response through a mechanism that is yet to be determined.

Hypomorphic mutations in the mammalian homolog of LTN1 cause neurodegeneration in mice (21). Similarly, mice with mutations in a central nervous system–specific isoform of tRNAArg and GTPBP2, a homolog of yeast Hbs1 which works with PELOTA/Dom34 to dissociate stalled 80S ribosomes, suffer from neurodegeneration (22). These observations reveal the consequences that ribosome stalls impose on the cellular economy. Eubacteria rescue stalled ribosomes with the transfer-messenger RNA (tmRNA)–SmpB system, which appends nascent chains with a unique C-terminal tag that targets the incomplete protein product for proteolysis (23). The mechanisms used by eukaryotes, which lack tmRNA, to recognize and rescue stalled ribosomes and their incomplete translation products have been unclear. The RQC—and Rqc2p’s CAT tail tagging mechanism in particular—bear both similarities and contrasts to the tmRNA trans-translation system. The evolutionary convergence upon distinct mechanisms for extending incomplete nascent chains at the C terminus argues for their importance in maintaining proteostasis. One advantage of tagging stalled chains is that it may distinguish them from normal translation products and facilitate their removal from the protein pool. An alternate, not mutually exclusive, possibility is that the extension serves to test the functional integrity of large ribosomal subunits, so that the cell can detect and dispose of defective large subunits that induce stalling.